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.