Appendage-resident epithelial cells expedite wound healing response in adult zebrafish.

Adult zebrafish are able to heal large-sized cutaneous wounds in hours with little to no scarring. This rapid re-epithelialization is crucial for preventing infection and jumpstarting the subsequent regeneration of damaged tissues. Despite significant progress in understanding this process, it remains unclear how vast numbers of epithelial cells are orchestrated on an organismic scale to ensure the timely closure of millimeter-sized wounds. Here, we report an unexpected role of adult zebrafish appendages (fins) in accelerating the re-epithelialization process. Through whole-body monitoring of single-cell dynamics in live animals, we found that fin-resident epithelial cells (FECs) are highly mobile and migrate to cover wounds in nearby body regions. Upon injury, FECs readily undergo organ-level mobilization, allowing for coverage of body surfaces of up to 4.78 mm2 in less than 8 h. Intriguingly, long-term fate-tracking experiments revealed that the migratory FECs are not short-lived at the wound site; instead, the cells can persist on the body surface for more than a year. Our experiments on "fin-less" and "fin-gaining" individuals demonstrated that the fin structures are not only capable of promoting rapid re-epithelialization but are also necessary for the process. We further found that fin-enriched extracellular matrix laminins promote the active migration of FECs by facilitating lamellipodia formation. These findings lead us to conclude that appendage structures in regenerative vertebrates, such as fins, may possess a previously unrecognized function beyond serving as locomotor organs. The appendages may also act as a massive reservoir of healing cells, which speed up wound closure and tissue repair.

Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish.

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.