Matrix metalloproteinases in osteoclasts of ontogenetic and regenerating zebrafish scales

Abstract

Matrix metalloproteinases (MMPs) are key enzymes in the turnover of extracellular matrix in health, disease, development and regeneration. We have studied zebrafish scale regeneration to ascertain the role of MMP-2 and MMP-9 in these processes. Scales were plucked from the surface of anaesthetised adult male zebrafish, and the scales that regenerated in the scale pocket were recovered at various time points after plucking. Analyses consisted of (i) mmp-9 in situ hybridisation; (ii) MMP-9+TRAcP double-staining; (iii) qRT-PCR for mmp-2 and mmp-9; (iv) zymography for gelatinolytic activity and (v) a hydroxyproline assay. We found that mmp-9 positive cells were confined to the episquamal side of the scales. Ontogenetic scales had irregular clusters of mono- and multinucleated mmp-9 expressing cells along their lateral margins and radii. During regeneration, mmp-9 positive cells were seen on the scale plate, but not along the lateral margins. Double staining for TRAcP and MMP-9 revealed the osteoclastic nature of these cells. During early scale regeneration, mmp-2 and mmp-9 transcripts increased in abundance in the scale, enzymatic MMP activity increased and collagen degradation was detected by means of hydroxyproline measurements. Near the end of regeneration, all of these parameters returned to the basal values seen in ontogenetic scales. These findings suggest that MMPs play an important role in remodelling of the scale plate during regeneration, and that this function resides in mononucleated and multinucleated osteoclasts which co-express TRAcP and mmp-9. Our findings suggest that the fish scale regeneration model may be a useful system in which to study the cells and mechanisms responsible for regeneration, development and skeletal remodelling.