Hepatocyte growth factor stimulates chemotactic response in mouse embryonic limb myogenic cells in vitro.

Abstract

In this study we investigate the influence of Hepatocyte Growth Factor (HGF) on the motility of embryonic forelimb myoblasts. Using Blindwell chemotactic chambers, it was found that HGF at concentrations of 1-50 ng/ml dramatically enhanced the ability of myogenic cells to migrate. This stimulatory effect was elicited in a dose-dependent fashion and the effect was reversed with the addition of HGF neutralizing antibodies. A checkerboard analysis was performed and it revealed that HGF's effect on limb myoblast motility was through both chemokinesis and chemotaxis. HGF was also examined for its ability to stimulate myogenic cell proliferation, using MF20 antibody as the myogenic marker. At all concentrations tested, HGF did not stimulate an overall increase in the numbers of MF20-positive myoblasts in culture. To examine the chemokinetic effect of HGF on cell migration in the limb, cells were isolated from the proximal regions of the limb (areas rich in myogenic cells), exposed to HGF, labeled with DiI and transplanted into 11.5 day mouse forelimbs. After 36 h of culture, it was found that DiI-labeled limb cells, pretreated with HGF, migrated significantly further in the limb than labeled cells that have not been exposed to HGF. The chemotactic effect of HGF was also investigated by implanting beads loaded with and without HGF into the 11.5 day limb. Proximal to the beads, DiI-labeled limb cells were also transplanted. It was found that HGF was able to chemotactically attract and direct the migration of DiI-labeled limb cells. Immunohistological staining was performed with HGF antibodies to determine the distribution of HGF in the 11.5 day mouse forelimb. It was found that HGF was strongly expressed by the apical ectodermal ridge (AER), the ectoderm and the mesenchyme directly beneath the AER. Positive staining was also obtained for the myogenic regions. However, the pattern was heterogeneous--punctuated with myogenic cells expressing and not expressing HGF.