It is well known that denervated amphibian limbs do not regenerate which has lead to the idea of limb regeneration being nerve-dependent. In fact, this nerve dependency for regeneration has been suggested not only for amphibian limbs but also for many other organs and structures in several regeneration models. However, and until now, very few neural factors have been shown to explain satisfactorily this nerve dependency. On the other side, the fact that it has been hypothesized in different models suggests that nerve dependency could be a common and specific feature for animal regeneration.
Now, a recent paper from the laboratory of Akira Satoh reports on the cooperative roles of Bmp and Fgf signalling pathways to induce limb formation in the absence of innervation in urodeles (http://www.ncbi.nlm.nih.gov/pubmed/25286122). The model they have used is the Accessory Limb Model (ALM). In this model, when a piece of skin is wounded and if, at the same time, a nerve is rerouted to the wound this wound can be induced to form a regenerative blastema, that does not grow into any patterned structure, and in fact it regresses over time. However, if together with this ectopic nerve supply a small piece of skin from the contralateral side of the wound is grafted into this wound, then the formation of an overall well-patterned ectopic limb is induced.
Previous studies have shown that when different Fgfs (fibroblast growth factors) are applied to skin wounds, an initial regenerative response with blastema formation can be induced, even in the absence of a nerve supply. However, an ectopic limb is not formed in these cases. So, Fgfs are not sufficient to induce a complete regenerative response. Here, the authors analysed the regeneration inductive function of Fgfs and Bmps in skin wounds. First, they found that Fgf2, Fgf8, Bmp2 and Bmp7 were expressed in dorsal root ganglion neurons and, therefore, can be considered as nerve factors. Then, the authors applied different combinations of these Fgfs and Bmps to see whether or not they could induce blastema and limb formation in skin wounds in the absence of nerve rerouting. The application of beads soaked either with Fgf2 and Fgf8 or Bmp7 alone was able to induce a blastema but not limb formation. However, when Bmp2+Fgf2+Fgf8 or Bmp7+Fgf2+Fgf8 were applied together, those induced blastemas were capable to keep growing and formed a patterned limb with digits, in most cases. Those induced blastemas showed normal expression of the blastema markers Prrx1 and Msx1 and the ectopic limbs, although missing some skeletal elements, showed an overall proper pattern with quite normal innervation.
The use of specific inhibitors of the Fgf or Bmp signalling pathways blocked limb formation in these skin wounds in which beads soaked with Bmp7+Fgf2+Fgf8 had been applied. Interestingly, in those cases, blastema formation was not inhibited indicating that single input of Fgf or Bmp signalling was sufficient to induce this blastema formation. However, simultaneous activation of both Fgf and Bmp pathways was necessary to induce limb formation. Next, the authors investigated whether Fgfs and Bmps were capable of inducing a blastema in denervated limbs. After denervation by removing the brachial plexus at the forelimb level and skin wounding and skin grafting, they applied beads with Bmp7+Fgf2+Fgf8. As a result they observed the formation of ectopic blastemas positive for Prrx1 and Msx2. Those blastemas, however, did not keep growing into a limb. The probable reason for that is that they were not innervated, suggesting that the later growing phase was dependent on axons attracted by the induced blastema cells.
Finally, the authors show that the use of Bmp2+Fgf2+Fgf8 or Bmp7+Fgf2+Fgf8 rescued also the regeneration ability in a denervated and amputated limb. Similarly to the rescue observed after skin wounding in denervated limbs, these factors induced blastema formation in denervated and amputated limbs, with a normal regenerative mitogenic response. Identical inductive effects of Bmps and Fgfs were seen when using the ALM model in the newt Pleurodeles waltl, indicating that these factors could work as general transformative agents from skin wound healing to limb formation in urodele amphibians.
Overall, this study further supports the idea of Fgfs and Bmps as neural factors that may explain, at least partly, nerve dependency of amphibian limb regeneration. In addition to an initial important role of those neural factors in blastema formation, the results obtained here also indicate that nerves may play important roles at later stages of limb formation. Thus, in denervated limbs Fgfs and Bmps induce blastema formation but these never grow into limbs as those blastemas do not get innervated. Further analyses those determine if these same Bmps and Fgfs or other nural factors are responsible of promoting blastema growth into a limb after axons are attracted from the stump region into the regenerative blastema.
The lack of gene-knockout technologies in many animal models of regeneration can be a problem to study gene function during this process. Using RNAi or morpholinos to produce knockdowns can somehow compensate these limitations. Recently, new methods of gene editing have been developed, including TALENs (transcriptional activator-like effector nucleases) and CRISPR (clustered regularly interspaced short palindromic repeat). Now, a recent paper from the laboratory of Elly Tanaka reports for the first time on the use of these novel technologies to study the effects of knocking out a Sox2 homologue during axolotl regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25241743).
As a first step to study gene deletion driven by TALENs and CRISPRs the authors tried to knock out a genomically inserted GFP-transgene in their strain of germline-transgenic GFP-axolotls. They injected the TALEN mRNAs or CRISPR RNAs into freshly laid embryos. Both methods turned out to be successful although CRISPRs appeared more efficient. Next, they used both methods to knockout an endogenous gene, a tyrosinase homologue. This gene is not essential for development and gives a pigmentation defect easily detectable. Again here, both methods worked although CRISPR-mediated knockouts were more penetrant and efficient.
Then, they moved to study the effects of knocking out Sox2, an important gene required for neural stem/progenitor cells maintenance and expansion in other animals, during both axolotl development and regeneration. Thus, they injected several different CRISPR RNAs into fertilized eggs at the single-cell stage and analysed them at 13 days post injection. Of 487 eggs injected with a particular Sox2-gRNA, 403 survived and grew up to normal sizes. Of those, 274 showed a curved body and many had excess blood in the olfactory bulb are. Also, they displayed a severe reduction of Sox2-positive cells in this olfactory bulb. Remarkably, and in contrast to mice in which Sox2 knockout is lethal, axolotls can apparently survive without this gene. To further corroborate these results, the authors knocked-down Sox2 using morpholinos and obtained a similar viability as for the CRISPR-mediated knockouts.
When analysing in more details the defects at the cellular level in the Sox2 knockout animals the authors found that although the expression of Sox2 in the cells lining the spinal cord lumen was lacking, those animals had an apparently normal organization of the spinal cord with normal NEU+ neurons, as well as normal expression of other neural stem cell markers such as GFAP and ZO-1 and the proliferation markers PCNA and phosphohistone H3. Then, the authors analysed the regenerative capabilities of these animals knockout for Sox2. To do that, they amputated the tail of those Sox2-CRISPR animals that showed a curved-body phenotype (and that were those that had a higher penetrance of deletions). Remarkably, Sox2 knockout axolotls showed reduced or lack of spinal cord in the regenerated tail. At day 6 of regeneration this reduction in the length of the regenerated spinal cord was not correlated with a shorter regenerated overall tail. By day 10, however, the Sox2 knockout animals also displayed a mild reduction of the overall length of the regenerated tail.
After a series of experiments with different markers the authors concluded that the deletion of Sox2 in neural stem cells resulted in a defect in the proliferative expansion of neural stem cells specifically after tail amputation. Compared to normal regenerating controls, it seems that in Sox2 knockout animals neural stem cells were not able to accelerate their cell cycle after amputation and a higher percentage of them appeared to remain in G1 or G2/M. Thus, the lack of Sox2 hampered proliferation and expansion of the neural stem/progenitor cell pool.
Finally, and with the aim of better understanding the different effects seen in embryogenesis and regeneration after knocking out Sox2, the authors analysed the expression of Sox3 because of the relationship between both genes in other models. Interestingly, here they found that during axolotl embryogenesis Sox3 showed indistinguishable expression patterns compared to Sox2, which would argue that Sox3 could compensate the lack of Sox2 during embryogenesis after Sox2-CRISPR knockout. On the contrary, during regeneration Sox3 was downregulated in the regenerating spinal cord, suggesting that the lack of Sox2 in those Sox2 knockout axolotls could not be compensated by the expression of Sox3.
In summary, this study shows that TALENs and CRISPRs can induce specific gene deletions in axolotls and be used to study regeneration in these animals at the genetic level. Remarkably, whereas Sox2 knockouts were viable and were developed with only mild morphological defects, these same animals failed to regenerate the spinal cord, revealing a regeneration-specific need for this gene in axolotls.
Sea cucumbers are well known for their ability to regenerate their digestive system as they can eviscerate and then regenerate their internal organs, including the gut. After evisceration the remaining mesenteries play a key role in gut regeneration. The laboratory of Jose García-Arrarás has published some reports describing how during regeneration an intestinal primordium develops from a thickening of the mesenterial edge. With time this thickening grows up to the formation of a tube that will become the regenerated gut. One of the components of the sea cucumber gut is the enteric nervous system (ENS) that innervates the gastrointestinal tract. Now, a recent paper from this same laboratory describes for the first time in detail the regeneration of the enteric ENS in the sea cucumber Holothuria glaberrima (http://onlinelibrary.wiley.com/doi/10.1002/reg2.15/abstract).
Despite some studies have reported that miss-function of the ENS can cause several neuropathies both neurodegenerative and inflammatory in mammals, very few studies have analyzed the regenerative potential of this system under those contexts. Here, the authors use the sea cucumber to study how the ENS regenerates de novo together with the new gut after evisceration. In a first set of experiments, the authors use a collection of antibodies to label specific neural projections and cells of this ENS. Although, as the authors point out, it is not clear what are the antigens recognized by these antibodies, they are useful to consistently label specific neural fibers and cell populations and see how this pattern is restored during regeneration.
The main components of the ENS were divided depending on their location within the mesothelial layer, the connective tissue and the luminal layer. In the mesothelium they found a fiber network within the muscle layer that innervates the visceral muscle. There is a second mesothelial plexus formed by large nerve bundles that run along the longitudinal axis of the gut, and mostly parallel to the longitudinal muscle fibers. Within the connective tissue there are thick fibers that would correspond to nerves connecting the mesothelial plexus with the connective and luminal layers. There is also a network of small neurons and fine fibers all throughout this connective layer. Finally, the luminal plexus is formed by neuroendocrine-like cells distributed among the luminal epithelial cells. Occasionally, some of these cells display fiber projections extending towards the mesothelium.
Here, the authors used their markers to follow the regeneration process of all these components of the ENS at 3, 5, 7, 10, 14, 21, 28 and 35 dpe (days post evisceration). Summarizing all their stainings ENS regeneration could be divided into several stages: 1) initially there was a neurodegeneration stage consisting in the degradation of the preexisting fibers within the mesentery edge that will give rise to the intestinal primordium (5-7 dpe); 2) during the first days, then, the intestinal primordium lacked any ENS innervation (8-10 dpe); 3) then, re-innervation of this primordium started by 14 dpe. Here, new fibers appeared, mainly proximally in the mesothelium from an extrinsic source, that is, from cells in the mesentery. Moreover, new cells (of intrinsic origin) appeared also within the connective tissue, also mainly in proximal regions; 4) the fourth stage at around 21 dpe, was characterized by the differentiation of large fibers crossing from the mesothelium to the connective tissue as well as for the intrinsic differentiation of new neural cells within the lumen epithelium; 5) by 28 dpe most of the mesothelium was innervated and no differences between proximal and distal areas were observed; finally 6) by 35 dpe the ENS pattern in the regenerated intestine was similar to normal non-eviscerated intestine.
It is important to point out that ENS regeneration occurred in parallel to other important events. Thus, for example, the initial neurodegeneration coincided with the remodeling of the extracellular matrix. Also, fiber regeneration coincided with myogenesis and the incoming neural fibers were possibly re-innervating the newly differentiated muscle fibers. An important question to answer in future experiments concerns the origin of the new ENS cells. The authors discuss that they could originate from the dedifferentiation of muscle cells or coelomic epithelial cells or, alternatively from enteric stem cells or glia present in the intestine or mesentery. In the case of the neuroendocrine cells they seemed to differentiate from the luminal epithelial cells.
In summary, the authors describe here the different stages that characterize the regeneration of the ENS in sea cucumbers. As some events are conserved in mammals following lesions or inflammatory responses as, for example, the observed degeneration-regeneration stages, H. glaberrima can be a good model to understand ENS plasticity in other models. Moreover, there is an obvious interest for bioengineers trying to obtain intestines for transplantation and, as the authors state, studying ENS regeneration could provide insights into the type of cells and timing at which ENS precursors should be added in order to make a properly functional intestine.
In amphibians, early steps for a successful regenerative response imply the de-differentiation of differentiated cell types and their re-entry into the cell cycle. A good example are newt myotubes that upon serum stimulation are induced to reprogram by dedifferentiating and re-entering the cell cycle, this last being dependent on the phosphorylation of Rb (retinoblastoma) and the downregulation of p53 activity. A recent paper from the laboratory of Maximina Yun and Jeremy Brockes analyses the role of ERK (extracellular signal-regulated kinase) signalling during newt myotubes reprogramming and how it may differ in muscle cells from regeneration-incompetent animals (http://www.ncbi.nlm.nih.gov/pubmed/25068118).
The first thing they saw was that serum stimulation of myotubes triggered a fast activation of ERK signalling that was sustained for up to 48 h. In addition to ERK, other MAPK pathways, such as JNK and p38, were also activated although at a much lower level. Then, the authors analysed whether the activation of those pathways was required for the re-entry to the cell cycle. By using different specific inhibitors of ERK, JNK and p38 alongside with serum stimulation, they found a differential disruption of Rb phosphorylation and cell cycle re-entry. The highest impairment was seen after ERK inhibition, which suggests that the activation of this pathway is critical for cell-cycle re-entry. The inhibition of ERK even at 24 h post serum stimulation impaired Rb phosphorylation suggesting that a sustained ERK activity is required for reprogramming.
Previous studies have shown that a sustained ERK activity results in the downregulation of Gadd45, a p53 target. Moreover, this same group has recently shown that the downregulation of p53 is a necessary step for newt myotube cell cycle re-entry. Here, a series of experiments combining ERK inhibition with p53 stabilization or inhibition suggests that the action of ERK signalling on cell cycle re-entry is mediated, at least in part, by downregulating p53 activity. This is further supported by the fact that ERK inhibition abrogated the downregulation of Gadd45 induced by serum stimulation. Next, the authors sought to determine whether ERK activity was also necessary to promote cell dedifferentiation in addition to cell cycle re-entry. To do this, they analysed the expression of Sox6, a muscle-specific gene. Upon serum stimulation the expression of this gene was downregulated, however, ERK inhibition abrogated this downregulation. Moreover, they also studied the effects of ERK inhibition on epigenetic changes that occurred in myotubes upon serum stimulation. The levels of expression of the repressive histone mark dimethyl H3K9 decreases upon serum stimulation. However, this decrease is abrogated by ERK inhibition. Taken into account that in other models it has been show that the demethylation of H3K9 is required for cell cycle progression and the expression of pluripotency-associated genes, the authors suggest here that ERK dependent-H3K9 demethylation in newt myotubes may provide a favorable environment for their reprogramming.
Finally, the authors compared these changes in ERK activity in newt myotubes with the response to serum stimulation of mouse myotubes. Upon serum stimulation, ERK was transiently activated in mouse myotubes at 1 h post induction, but then the levels went down to baseline after 3 h. In addition, no changes in the repression marker dimethyl H3K9 were observed. These results suggest that the extent of ERK signalling could underlie differences in the regenerative capabilities shown by salamander and mammalian cells.
In summary, the authors propose here that a sustained activation of ERK signalling leads to the downregulation of p53 activity, which would facilitate cell cycle re-entry through Rb phosphorylation as well as alterations in the gene expression landscape facilitating also cell dedifferentiation. Future experiment should try to determine the upstream tyrosine kinase receptor that activates ERK as well as the serum component responsible of such activation, and subsequent reprogramming of newt myotubes.
One of the many amazing features of animal regeneration is that although broadly distributed throughout phylogeny, there is an enormous heterogeneity in the regenerative capabilities shown by closely related species. A typical example is the capacity shown by some vertebrates to regenerate their limbs. Whereas many amphibians (newts, axolotls, frogs) are able to regenerate their limbs and tails and some fishes regenerate their fins, mammals have lost this ability. This heterogeneity has raised the question whether regeneration was a basal condition at the root of animal evolution and has been subsequently lost in some lineages or, alternatively, it has appeared independently in some animal groups. The fact that many regenerative models share a wide number of features and conserved signalling and genetic pathways controlling key aspects of the regenerative process supports the homology of animal regeneration. On the other hand, some studies have reported the existence of salamander-specific genes required for regeneration, which has been used to propose that the regenerative abilities may have appeared independently in different lineages. However, despite that some specific features may exist in each of the current regenerative models it is also evident that most of them share many more other key properties of regeneration. Somehow it is similar to considering embryogenesis as a basal conserved feature (with the dozens of conserved genes and pathways playing homologous roles) that has acquired some specific traits in different species depending on the type of fecundation or type of egg, among others.
Going back to the case of limb regeneration in tetrapods, a recent paper from the laboratory of Nadia Fröbisch reports on evidences of limb regeneration in a 300-million-year-old-amphibian (http://www.ncbi.nlm.nih.gov/pubmed/25253458). These observations have been made in several very well preserved specimens of Micromelerpeton crederni, a basal member of the dissorophoid clade within the temnospondyl amphibians. Although the phylogenetic position of modern amphibians remains still under debate, the authors state here that most scientists consider that dissophoroid temnospondyls including Micromelerpton represent the stem lineage of modern amphibians.
Many current amphibians are able to regenerate their limbs in a very precise way, so the regenerated limb is undistinguishable from the original one. However, there are also cases in which such regeneration is not perfect and some abnormalities appear. These cases may include repetitive amputations of the limbs, interference of some key early steps of regeneration or amputation at different stages of the life cycle. What it has been shown is that the abnormalities that appear during limb regeneration are usually different from the abnormalities that normally appear during limb development. What the authors of this study have found is that the pattern and combination of abnormalities in the limbs of the Micromelerpeton fossils are directly comparable to the variant morphological patterns in the regenerated limbs of current salamanders. These patterns include fusions along the proximo-distal axis and abnormalities predominantly located on the preaxial side of the autopods. Thus, the most common variant caused by abnormal regeneration in salamanders is an increase or decrease in the count of phalangeal numbers, which is also the most frequently abnormality in Micromelerpteon fossils.
In summary, the results presented here suggest that Micromelerpteon was capable of regenerating its limbs further suggesting that limb regeneration was an ancient capacity of the dissorophoid lineage leading towards modern amphibians and that has been retained in some lineages (such as salamanders). Further studies should try to determine the causes that have lead to the maintenance or loss of such regenerative ability in the dissorophoid lineage.
There has been a high level of evolutionary conservation in the function that several signaling pathways play during regeneration. Thus, it is well known the role that pathways such us BMP/TGF-b, Wnt/b-catenin, notch, Hedgehog and RTKs have during the regeneration of different structures in a variety of animals from axolotls and zebrafish to Hydra and planarians. There are some cases in which the ability to regenerate in different species depends on different tissue dependencies. An example is the regeneration of the tail in axolotls and Xenopus tadpoles. In axolotls the spinal cord is necessary for tail regeneration, whereas in Xenopus tadpoles is the notochord and not the spinal cord what is required. In axolotls this dependency seems to be determined by Hedgehog signaling as shh (sonic hedgehog) is exclusively expressed in the spinal cord. Now, a recent paper from the laboratory of Yuka Taniguchi and Makoto Mochii reports that in Xenopus tadpoles shh is expressed in the notochord and required for tail regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24941877).
Whereas in axolotls shh is expressed in the spinal cord during tail regeneration, the authors show here that shh was exclusively expressed in the notochord in the entire regeneration region in Xenopus tadpoles. On the other side, shh receptors patched 1 and 2 (ptc-1 and ptc-2) were expressed in the spinal cord in them. In order to determine the function of Hh signaling on tail regeneration the authors used cyclopamine a widely used inhibitor for Hh signaling. Upon cyclopamine treatment tail regeneration was severely impaired as well as the length of the regenerating notochord. These effects were rescued by the treatment of pumorphamine, an agonist for the Hh pathway. Cyclopamine treatment did not result in an increase in apoptotic cells, but a significant down-regulation of genes related to the Hh pathway such as ptc-1, ptc-2, gli-1 and smo was observed in those treated animals.
During normal tail regeneration, undifferentiated notochord cells accumulate at the distal edge of the amputated notochord by day 2. Then these cells align perpendicular respect to the AP axis and differentiate into cells containing large vacuoles after day 3. In contrast, upon cyclopamine treatment, undifferentiated notochord cells normally accumulated at the edge of the amputated structure and proliferated; however, their posterior alignment and final differentiation was inhibited. Interestingly, cyclopamine treatment impaired the growth of the regenerating spinal cord as well as the formation of myofibers. In fact, the expression of myoD, a well-known myogenic transcription factor, was suppressed upon treatment. Also, the authors observed a strong reduction in the number of cells positive for Pax-7, another myogenic marker. These results suggest a strong dependence of muscle regeneration on Hh signaling, being this in agreement with previous results in mouse and chicken in which shh has a positive effect on the proliferation and differentiation of the satellite cells (muscle stem cells).
Overall, the results presented here uncover a pivotal role of shh in tail regeneration in Xenopus tadpoles as its inhibition impairs regeneration by affecting several processes such as the final differentiation of new notochord cells as well as the proliferation of progenitors for the spinal cord and muscle fibers. Future experiments should determine whether the defects observed here in the differentiation of the notochord are due to a direct autocrine action of shh (as it is expressed in the notochord) or an indirect effect of shh function on spinal cord regeneration.
In summary, this work describes how the differential expression of shh either in the notochord or the spinal cord could account for the different tissue dependency of tail regeneration in Xenopus tadpoles and axolotls, respectively.
Back from the 2014 EMBO Conference on The Molecular & Cellular Basis of Regeneration & Tissue Repair held at St. Feliu de Guixols. This was an excellent meeting mainly focussed on basic aspects of regeneration in several animal models. The role of ROS signalling and the inflammatory response on wound healing and the triggering of a regenerative response were some of the hottest topics of the meeting, as well as neural and heart regeneration. Also, there were some talks and posters on new (or not so popular yet) models for regeneration such as the cnidarian Nematostella vectensis, several echinoderms and the crustacean Parhyale hawaiensis. It is also clear that large transcriptomic analyses and the enormous amount of data currently generated from RNAseq experiments in all different models and regenerative contexts will help to identify the molecular and cellular bases of key events of regeneration such us wound healing, blastema formation and growth, patterning and polarity, among others. Thus, comparative analyses might uncover novel conserved and taxon-specific elements required for regeneration. I hope we can discuss all this data in the near future in this blog.
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 (http://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.
Just a short notice to inform you that I am going to take some weeks off for summer break. I will back in September to continue posting about regeneration. The beginning of September will be also an exciting time as there will be the EMBO Conference on Regeneration in Sant Feliu de Guixols (near Barcelona), so I hope we can learn a lot from the data presented there.
Have a nice summer!