Several weeks ago I commented on a study from the laboratory of Gilbert Weidinberg in which they had characterized an organizing center defined by the Wnt/b-catenin pathway within the distal blastema of regenerating zebrafish fin, that would control regeneration by regulating the function of several downstream signaling pathways that would mediate the effects of this organizer on surrounding tissues. Here, I comment on a study from the laboratory of Scott Stewart and Kryn Stankunas that describe how the Wnt/b-catenin and BMP signaling pathways work together and in opposite directions to coordinate bone regeneration during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24485659).
In zebrafish, bone regenerates through dedifferentiation and re-differentiation of lineage-restricted osteoblasts. Osteoblasts are the responsible of depositing the osteoid, a unique extracellular matrix that form the mature bone. Although previous reports have implicated several signaling pathways, including Wnt/b-catenin and BMP receptor, in this process, how they act at the cellular and molecular levels to drive a successful regeneration is not completely known. Here, the authors first analyzed the expression of Runx2 and sp7, two transcription factors with well-known roles on bone formation. Early after amputation Runx2 was upregulated in osteoblasts lining preexisting bone adjacent to the amputation plane. Later, some Runx2+ mesenchymal cells expressed also sp7. Then, Runx2-/sp7+ cells first appeared near the amputation plane. By 72h of regeneration the osteoblast lineage was highly organized along the proximo-distal axis of the blastema: Runx2+ cells were located in most distal regions while sp7+ cells were mainly found near the amputation plane. In between Runx2+/sp7+ cells were found. In terms of proliferation, more Runx2+ cells incorporated EdU compared to sp7+ cells, suggesting that sp7+ cells near the amputation plane would be non-proliferative osteoblasts that append to progressively elongating bone.
After amputation, osteoblasts dedifferentiate to give rise to Runx2+ preosteoblasts. The authors showed that osteoblasts have epithelial-like properties as they were labeled with antibodies against catenins (a- and b-) that are found in adherens junctions that interconnect epithelial sheets. During regeneration, distal Runx2+ did not express a-catenin in contrast to Runx2+/sp7+ and sp7+ cells in close proximity to new bone, that were positive for membrane-localized a-catenin. At 24h after amputation osteoblasts rapidly lost a-catenin expression as they dedifferentiate into a progenitor state. Moreover, as they became Runx2+ and Runx2+/sp7+ cells they changed their shape from long and thin to a more compact, polygonal morphology. These results suggested that osteoblast went through an epithelial-to-mesenchymal transformation (EMT) during regeneration. This was further supported by the observation that twist2, a well-known transcription factor that directs EMT, and runx2a were rapidly induced in tissue adjacent to the amputation plane. Later, distal Runx2+ cells co-expressed twist2. Therefore, the authors concluded that Runx2+ cells originated from EMT of differentiated osteoblasts and distal Runx2+ preosteoblasts were maintained in a mesenchymal twist2-expressing state.
Next, the authors analyzed the Wnt/b-catenin signaling in regenerating fins. At 24h of regeneration, Runx2+ cells had nuclear b-catenin staining. By 72h post amputation, strong nuclear b-catenin was observed in distal Runx2+ preosteoblasts with much less staining in sp7+ differentiating osteoblasts near the amputation plane, suggesting that downregulation of Wnt signaling correlates to osteoblast maturation. It is known that Wnt signaling can initiate EMT and induce twist expression during mouse bone development. Here, the authors used IWP-2, an inhibitor of Wnt signaling. IWP-2 treatment arrested regeneration by interfering with osteoblast EMT and the induction of twist2 expression, indicating an important role of Wnt signaling in osteoblast EMT. Moreover, IPW-2 treatment from 48h to 72h post-amputation also blocked regeneration by depleting osteoblast-lineage cells distal to the amputation plane, suggesting a role of this pathway in maintaining the preosteoblast population.
Finally, the authors analyzed the BMP pathway as it has been also implicated in bone formation. The activation of the BMP signaling leads to the phosphorylation of the transcription factor Smad1/5/8, that can then go to the nucleus and activate its downstream target genes. Here, pSmad1/5/8 was detected in differentiating sp7+ cells but not in Runx2+ or Runx2+/sp7+ preosteoblasts. The inhibition of BMPR resulted in a pronounce decrease in the extend and levels of sp7 expression and reduced bone formation. These treated fins were able to form a blastema but failed to produce mineralized bone, suggesting a role of the BMP pathway in osteoblast maturation. Remarkably, BMPR inhibition resulted also in an increase in the number of Runx2+ cells and a decrease of the Runx2+/sp7+ and sp7+ populations. Osteoblast proliferation and cell death were not affected by this treatment suggesting that BMP would drive osteoblast differentiation.
As BMP inhibition expanded proximally the distal Runx2+ population the authors hypothesized that BMP activity in proximal regions would normally inhibit Wnt/b-catenin activity in those proximal domains. This was supported by the observation that BMPR inhibition reduced the expression of Dkk proteins, well-known negative regulators of Wnt activity. In agreement with the idea of distal Wnt active and proximal BMP active populations, wnt5a and wnt5b were mainly expressed at the distal tip of the blastema whereas bmp2 was expressed in differentiating proximal osteoblasts.
In summary, the authors have shown that zebrafish bone regeneration is mainly regulated by the antagonistic and coordinated function of the Wnt and BMP signaling pathways in order to provide a precise balance between cell plasticity and differentiation. In their proposed model, Wnt activity drives EMT of osteoblasts to give rise to dedifferentiated Runx2+ preosteoblasts. Sustained levels of Wnt activity in the distal blastema maintain these Runx2+ proliferative cells. Then, as these preosteoblasts are located to more proximal regions they upregulate bmp2 and activate autocrine BMP activity that promotes osteoblast differentiation by inducing the expression sp7 and dkk1b, that inhibits Wnt activity to prevent the overexpansion of the progenitor pool.
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.
Wound healing is a universal response to injury conserved in all animals. However, not in all cases wound healing is followed by a successful functional regeneration. Thus, for example, skin injuries in adult mammals are usually solved through a so-called scarring wound healing that does not allow a functional recovery of the damaged skin. On the other side, in regeneration-competent species wound healing does not a have a negative effect but on the contrary is a key first step to trigger a regenerative response. Thus, in those cases, impairing wound healing results in the inhibition of regeneration. A deep characterization of the cellular and molecular events that result in scarring or regenerative wound healing may be very important to try to develop strategies and therapies to enhance the poor regenerative abilities shown by many animals. Now, a recent paper from the laboratory of Mark Martindale has characterized the regenerative wound healing in the cnidarian Nematostella vectensis (http://www.ncbi.nlm.nih.gov/pubmed/24670243).
In a first set of experiments the authors characterized the cellular and molecular events that occurred after injuring the animals by making punctures in their bodies with a glass needle. Two hours after injury an enrichment of actin was seen around the injury site and the wounds were healed after 6 hours. In another cnidarian, Hydra, and some vertebrates, apoptosis is required to trigger a proliferative response that leads to a successful regeneration. Similarly, upon injury along the ectodermal surface of Nematostella, apoptosis was significantly upregulated. Next, the authors decided to conduct a pharmacological screen to see which signaling pathways could have a role in wound healing and regeneration in these animals. Inhibition of the Notch pathway blocked head regeneration without affecting wound healing. On the other side, and unexpectedly, they did not found any defect after blocking the TGFB signaling. Finally, they inhibited ERK signaling and found a strong impairment of wound healing and regeneration. The MAPK signaling pathway plays many functions including immune response, cell proliferation, apoptosis and cell movement. In Drosophila, ERK (through MAPK) regulates actin dynamics at the injury site a the early stages of wound healing. Using their puncture assay they found that inhibiting ERK signaling with the drug U0126 caused wound to remain open after six hours and also eliminated the local phosphorylation of ERK at one hour after injury, compared to the wound response of untreated animals. U0216 did not blocked the initial apoptotic response to injury indicating that apoptosis by itself is not sufficient to trigger a regenerative response. Also, the animals treated with U0216 did not show much actin concentration around the injury suggesting that ERK could be targeting cell movement and adhesion.
Then, the authors used Nematostella genome-wide microarrays to identify genes involved in wound healing. They analyzed the gene profiles from samples taken 1 hour and 4 hours after injury in untreated and U0216 treated animals, which allowed them to do many comparisons. Thus, they generated a profile of genes not only up- or down-regulated at early (1h) and late (4h) stages of normal wound healing, but also how the expression of those genes was affected after inhibiting the ERK pathway. After injury and wound healing genes upregulated included genes with peptidase activity, modulators of MAPK signaling, Sox E1 and runt transcription factors, growth factor-related genes as well as genes related to mucus proteins. Some of these genes were validated by qPCR and/or in situ hybridizations. The authors focused then in several genes: uromodulin, soxE, thiamine enzyme, a matrix metalloproteinase (MMP) inhibitor and a maltase-like gene. In all cases these genes were upregulated upon injury and this upregulation appeared dependent of ERK signaling, as it was not observed after treatment with U0216. Remarkably, all these genes were upregulated during regeneration after amputation through the oral-aboral axis. Again, the expression of these genes during regeneration was dependent of ERK signaling.
To conclude, the authors propose that ERK signaling would be necessary for the initiation of the early wound healing response in Nematostella, agreeing with the important functions of the ERK signaling during regeneration reported in other systems. Future functional analyses on the genes identified here should help to confirm this hypothesis and to better characterize wound healing at the gene expression level. In summary, this is the first report of genes involved in wound healing in Nematostella. Comparisons of the cellular and molecular events that characterize Nematostella wound healing with those found in other regenerative models as well as in regeneration-incompetent animals could help to understand better this key initial process that takes place after any injury.