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.

Skin healing and scale regeneration in fed and unfed sea bream, Sparus auratus

“…sonic hedgehog (SHH) and 2) the timescale of the experiment was not ideal to identify changes in gene expression of developmental genes associated with skin healing since by day 3 histological analysis revealed that epidermis is already re-established. Furthermore, metalloproteinases 2 (MMP-2) and 9 (MMP-9) have been recently suggested to have a role in scale regeneration in zebrafish [21]. However, MMP-2 is not represented in the sea bream microarray and although MMP-9 is represented (SAPD23115) it did not change significantly between the groups analyzed.…”

“…The ectodysplasin-A-receptor (EDAR) has been shown to be required for scale initiation and may also be involved in the cross-talk between the epidermal basal cells and the differentiating scale-forming cells in medaka ( Oryzias latipes ) [20]. Moreover, recently MMP-2 and MMP-9 were shown to have a role in scale regeneration in zebrafish [21]. Removal of scales damages a key barrier of the innate immune system and consequently provokes an inflammatory response and activation of the processes associated with healing and skin and scale re-growth [22,23].…”

Evolution of the angiopoietin-like gene family in teleosts and their role in skin regeneration

“…Skin and scale regeneration in fish involves, re-epithelialization and differentiation of scale-forming cells (day 1–2), production of the external layer matrix (days 3–5), production of the basal-plate matrix (days 6–14) and finally partial mineralization of the basal plate (days 14–28) [12]. Wound repair and skin regeneration studies are numerous in mammals [13, 14] and amphibians [15, 16], but are much less frequent in the fishes, the largest group of extant vertebrates [17] and the molecular basis of skin repair is generally restricted to single gene studies [18–21]. Recent studies have used microarrays to assess the response of fish skin to damage or ectoparasites [8, 22] and members of the angiopoietin family are among the differentially expressed genes detected.…”

Use of zebrafish models to investigate rare human disease

“…They discovered that atp6v1h deficiency results in elevated transcript levels of the osteoclast marker, matrix metalloproteinase-9 ( mmp9 ). This was also the case for mmp13a , which is involved in mmp9 -mediated control of bone homeostasis 81. Corroboration of these findings came through the demonstration that murine Mmp9 and Mmp13 are also upregulated on Atp6v1h knockdown in mouse osteoclasts.…”