Matrix metalloproteinases and collective cell migration in 24 h primary zebrafish explant cultures: MMP13 plays an inhibitory role and MMP14 may respond to stretch during reepithelialisation

Abstract

Zebrafish keratocytes collectively migrate rapidly when established in explant cultures but little is known about the signals that initiate motility or the signal transduction pathways that result in an epithelial to mesenchymal transition. Matrix metalloproteinases (MMPs) are strong candidates for playing a role in this regulation and have previously not been analysed in this wound healing model system. Results presented here document a rapid and dramatic rise in MMP14a, MMP2, MMP9 and MMP13a mRNA levels over time. In a motility assay, a broad-spectrum MMP inhibitor and an inhibitor specific for MMP2 and MMP9 significantly decrease cell migration in a dose dependent manner but treatment with an MMP13 specific inhibitor significantly increases cell sheet area. Immunofluorescence staining with an antibody specific for the catalytic domain of MMP14 indicates that activated MMP14 protein is highly expressed on cells at the leading edge of a sheet compared with follower cells in the centre of the sheet, and is augmented further in leader cells that are stretched, thus likely in the process of detaching from the cell sheet. These data are consistent with a model in which active MMP14 at the leading edge of cell sheets in explant cultures triggers activation of MMP2 and/or MMP9, thus creating promigratory signal(s) that outweigh the inhibitory role of targets cleaved by MMP13. Taken together, these data suggest that MMPs play an important but complex role in regulating the collective cell migration of zebrafish keratocytes and provide support for the relevance of using zebrafish as a model for human disease.