In previous posts I have discussed the important role of macrophages and the immune system in triggering a successful regeneration in contrast to a scarring wound healing, by modulating the inflammatory response. Thus, for example, I commented on the requirement of macrophages to induce blastema formation during limb regeneration in salamanders probably by activating cell dedifferentiation and the proliferation of progenitor cells needed to rebuild the missing tissues (https://regenerationinnature.wordpress.com/2013/06/06/macrophages-and-limb-regeneration-in-salamanders/). Now, a paper from the laboratory of Timothy Petrie and Randall T. Moon reports on the need of macrophages also during tail regeneration in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24961798).
Zebrafish tail regeneration may be divided into three main stages: 1) wound healing (0-1 days of regeneration); 2) blastema formation (1-3 days of regeneration); and 3) regenerative outgrowth and patterning of the new tissue (>3 days of regeneration). Previous studies in zebrafish larvae had reported that upon injury neutrophils and macrophages accumulate at the wound region suggesting a similar role of these cells compared to their mammalian counterparts. However, the role of these inflammatory cells in adult zebrafish regeneration remains mainly unknown.
In this study, the authors used several transgenic lines to track neutrophils and macrophages upon amputation. For neutrophils, these cells rapidly accumulated at the wound from 6 hours post amputation (hpa). A maximum peak was achieved by 3 days post amputation (dpa) and then their number declined from 5 dpa until reaching pre-amputation levels of neutrophils by 7 dpa. It is known that regeneration rates are different along the proximo-distal axis of the tail. Thus, proximal amputations result in faster regeneration compared to slower growth upon distal amputations. Interestingly, proximal amputations recruited over twice the number of neutrophils as distal ones. Whereas few neutrophils were detected in uninjured tails, macrophages were found in higher density. Upon amputation macrophages began accumulating in the wound region by 3-4 dpa, reaching a peak by 6-8 dpa. As neutrophils, macrophages also accumulated faster and at greater densities in proximal amputations.
Upon injury, neutrophils accumulation appeared to depend on their migration from the vasculature near the wound. The authors inhibited neutrophil recruitment into the wound but this reduced accumulation did not result in any difference in the rate of regeneration compared to controls. This is in contrast to what happens in larval tails as previous reports have suggested that neutrophil deficiency increases the regeneration rates. Next, the authors sought to determine the consequences of macrophage ablation during regeneration. They used a transgenic line bearing the enzyme nitroreductase (NTR) downstream of a macrophage-specific promoter. NTR converts the pro-drug metronidazole (MTZ) into a cytotoxic agent that kills the cell. Upon 36 h of MTZ treatment there was a strong reduction of around 80-90% of the macrophages in the tail. In control animals, MTZ by itself did not affect either the inflammatory response or the regeneration rate and success. Then, the authors amputated the tail and treated the animals with MTZ for 14 dpa. They saw that macrophage ablation resulted in a significant decrease of the extent of new tissue growth. Moreover, these fish also showed aberrant tissue growth along the regenerated tail. The zebrafish tail is composed, among other tissues, of segmented bony rays. Macrophage ablation resulted in a reduction in the average number of segments in the regenerated ray. Also, the degree of mineralization was reduced in NTR+MTZ fish, indicating that macrophage depletion impaired bone ray patterning and the quality of bone formation.
Then, the authors analyzed in more detail which events required for regeneration could be affected upon macrophage ablation. They observed that although the loss of macrophages did not significantly affect gross blastema morphology and size, there was a significant decrease in cell proliferation. Also, at 4 dpa there was a strong reduction in the expression of regeneration-associated and injury-response genes. On the other hand, neutrophils normally accumulated upon macrophage ablation. In order to determine at what stage of regeneration are macrophages required the authors ablated them at two distinct time points. In one set of experiments they ablated them from 2 days before amputation through 3 dpa, corresponding to the stages of wound healing and blastema formation. The authors observed the same defects on regenerative rate and aberrant morphologies of the regenerated tails as when MTZ treatment was applied for 14 dpa. On the other side, and in order to analyze the requirement of macrophages during the outgrowth stage, the authors ablated the macrophages from 3 dpa through 14 dpa. In those experiments, the regeneration rate was not significantly affected; however, they still observed a higher occurrence of aberrant morphologies of the regenerated tails. So, it seems that during the early stages of regeneration macrophages would be required for blastema formation, whereas during tissue outgrowth macrophages would be also required to modulate tissue patterning.
Finally, the authors wanted to study the relationship between the Wnt/b-catenin pathway and inflammation during tail regeneration as it has been shown that this pathway is required for blastema formation and outgrowth in zebrafish tail regeneration. Moreover, Wnt/b-catenin modulates several inflammatory processes in other models. Again, they used several transgenic lines to track the activation of this signaling pathway. As described above for neutrophils and macrophages, a greater density of cells with activated Wnt/b-catenin signaling were found in proximal amputations compared to distal ones. Flow cytometry analyses showed that less than 1% of neutrophils and 3% of macrophages exhibited activated Wnt/b-catenin signaling. Interestingly, macrophage accumulation at the wound was almost completely inhibited after inhibiting Wnt/b-catenin. Also, the inhibition of this pathway resulted in delayed neutrophil resolution and prolonged neutrophil number in the wound region, suggesting that the Wnt/b-catenin pathway might be required for the progression of the injury response after amputation. Thus, Wnt signaling might mitigate the initial inflammatory response and function as a molecular switch from neutrophil resolution to macrophage enrichment.
In summary, this study reports on the requirement of macrophages for zebrafish tail regeneration providing a functional link between inflammation and regeneration.