Effect of platelet-derived growth factor isoforms on the migration of mouse embryo limb myogenic cells

The effect of platelet-derived growth factor (PDGF) isoforms on limb myoblast migration was examined in vitro. Using Blindwell Chemotaxis chambers, the ability of PDGF-AA, -AB and -BB to stimulate the migration of myoblasts, obtained from the proximal region of 11.5 day mouse forelimb buds, was examined. Immunocytochemistry, with the anti-sarcomeric myosin antibody MF-20, was used to identify the myogenic cells in the heterogeneous cell population. Myoblasts, suspended in PDGF-free medium in the upper chamber, migrated across the polycarbonate filter of the Blindwell chamber to 1-10 mg/ml PDGF-AB and 1-100 ng/ml PDGF-BB situated in the lower well. At 1-10 ng/ml of either PDGF-AB or -BB migration increased in a dose-dependent manner. PDGF-AA, however, was unable to elicit a significant locomotory response in forelimb myoblasts. A Checkerboard assay, with various concentrations of PDGF-AA,-AB or-BB in the upper and lower wells of the chamber, indicated that -AB and -BB but not -AA stimulated the random migration of limb myoblasts. The differential effect of PDGF isoforms on myoblast migration was compared with other aspects of skeletal muscle development. At 0.1-10 ng/ml all three isoforms were able to stimulate an increase in the number of differentiated myoblasts, indicated by the expression of sarcomeric myosin, on examination after 48 h when cultured at low density. In high density cultures, however, these isoforms inhibited myoblast fusion when compared to the spontaneous fusion observed n untreated cultures. Immunohistochemical studies of both cultured limb cells and cryosections of 11.5 day whole limbs revealed that myoblasts expressed both PDGFα-and β-receptors which suggests that the action of PDGF isoforms on limb myoblasts is receptor-mediated. Finally, having demonstrated that the PDGF-B monomer stimulates migration in limb myoblasts, by immunohistochemistry, the presence of PDGF-B was confirmed and its distribution examined in the 11.5 day forelimb.