In recent years several papers have uncovered the importance of ion channels during regeneration in different models. Thus, for example, cellular hyperpolarization is essential for Xenopus tadpole tail regeneration and cellular depolarization is required to specify anterior polarity in planarians. Now a recent paper from the laboratories of Ana Certal and Joaquín Rodríguez-León has reported for the first time the requirement of V-ATPase activity for fin regeneration in adult zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24671205).
After any wounding an electric current is generated as a response; however, only in those cases in which a regenerative process is triggered these endogenous electric currents are maintained beyond wound closure. Here, the authors first analysed the contribution of different ions (K+, Na+, H+, Ca2+ and Cl–) to the electric current during adult zebrafish fin regeneration. Of all these ions H+ was the only one for which the authors found that 24 hpa (hours post-amputation), during blastema formation, there was still an efflux that was 14-fold higher than the one detected in intact fins. Previous microarray experiments had detected V-ATPase, a main H+ transporter, as being upregulated after 24h during fin regeneration. Thus, the authors checked the expression of several V-ATPase subunits during regeneration. Two of them, atp6v1a and atp6ve1b, were not expressed in intact fins but were upregulated in the blastema by 24 hpa. At 72 hpa some expression was still detected at the distal part of the blastema. Next, the authors assessed the role of V-ATPase during regeneration. They blocked the pump’s activity either by using concA or morpholinos (MO) against atp6v1e1b. Both approaches delayed fin regeneration suggesting a role for this H+ pump in the regenerative process.
As it happens for amphibian limb regeneration, proximal amputations of the caudal fin resulted in higher regeneration rates compared to distal stumps. Interestingly, the expression of atp6v1e1b was already visible by 12 hpa, whereas in distal stumps the first expression of this gene was observed at 24 hpa. Not only the expression appeared earlier but it also covered a wider region. By 48 hpa these differences were not so obvious anymore. In agreement with this earlier and stronger upregulation of atp6v1e1b in proximal stumps, the authors found out that the H+ efflux started earlier in those proximal stumps (3 hpa instead of 12 hpa in distal ones) and was higher at any time point measured than in distal stumps. These results clearly indicate a relationship between V-ATPase and H+ efflux and the regeneration rate along the PD (proximo-distal) axis. Further supporting this, atp6v1e1b knockdown significantly decreased the H+ efflux. This MO-mediated silencing of atp6v1e1b also resulted in a decreased regenerated area, being this inhibition higher in proximal stumps, suggesting that those proximal stumps with higher regenerative rates are more dependent on V-ATPase activity. Remarkably, the inhibition of V-ATPase activity did not seem to affect the regeneration of the larval fin fold.
Finally, the authors studied the effects of inhibiting the V-ATPase activity on cell proliferation and gene expression. Although no differences in proliferation were observed at 24 hpa, by 48 hpa atp6v1e1b knockdowns showed a significant reduced number of proliferative cells within the blastema, compared to controls. Different signalling pathways, including FGF, Wnt/B-catenin and Retinoic acid (RA), have been shown to regulate cell proliferation during regeneration. In controls, the expression of mkp3 (FGF signalling) and aldh1a2 (RA signalling) was detected in wider domains in proximal stumps compared to distal ones, similarly to the differences observed for V-ATPase activity. The silencing of atp6v1e1b resulted in the inhibition of the expression of mkp3 and aldh1a2 indicating that V-ATPase was required for the expression of these two genes during regeneration. Last, the authors reported that V-ATPase seems to be also necessary for the normal innervation of the regenerating fin.
In summary, this study reports for the first time the requirement of V-ATPase for adult zebrafish fin regeneration. The authors propose that the regulated H+ efflux generates pH and/or voltage domains within the regenerating tissue that, directly or indirectly (for example, via innervation), would act on FGF and RA signalling pathways to regulate cell proliferation during regeneration.