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

Abstract

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 ng/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 in untreated cultures. Immunohistochemical studies of both cultured limb cells and cryosections of 11.5 day whole limbs revealed that myoblasts expressed both PDGF alpha- and beta-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.