regeneration in nature

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Senescence and regeneration in salamanders

Cellular senescence is a state in which cells stop dividing but remain metabolically active. Moreover, they usually express a pro-inflammatory secretome, upregulate immune ligands and are positive for specific activities such as senescence-associated b-galactosidase (SA-bgal). Cells can enter senescence after DNA damage and activation of oncogenes, for example, to prevent cell proliferation. Recently, senescence has been linked to aging and aged-related pathologies, as in many species senescent cells accumulate with aging. In mammals, our limited regenerative abilities are further compromised with age. A recent report indicates that the decline in muscle regeneration with age could be related to an increase of cellular senescence. But what happens in those other vertebrates such as amphibians capable of regenerating repetitively over most part of their lifespan? Very few studies have addressed the regulation of senescence and its relationship with regeneration in those regeneration-competent species. Now, a recent paper from the laboratory of Maximina Yun (http://www.ncbi.nlm.nih.gov/pubmed/25942455) reports on cellular senescence during amphibian limb regeneration.

First, the authors set up a system to identify senescent cells in cell culture and tissue sections in salamanders. As senescence stops the proliferation of damaged or dysfunctional cells they use UV irradiation to induce DNA damage leading to senescence. Twelve days after irradiation about 80% of newt A1 cells entered a senescent state characterized, among others, by high levels of SA-bgal activity, sustained production of reactive oxygen species (ROS) and extended mitochondrial and lysosomal networks. Also, they acquired a secretory phenotype. In contrast to quiescent cells, these senescence ones did not re-enter the cell cycle upon serum stimulation. All these traits observed in salamander senescent cells were comparable to those previously observed in mammalian cells.

Next the authors analysed cellular senescence in vivo in normal and regenerating newts. During regeneration they observed a significant induction of cellular senescence during the intermediate stages of this process. However, the number of senescent cells decreased at later stages. Senescent cells were found at the amputation plane and within the blastema and included different cell types such as cartilage, muscle, fibroblasts and epidermal glands. Similar results were observed in axolotls, another amphibian model for regeneration. Remarkably, this induction and posterior disappearance of senescent cells was specific of regeneration, as it was not observed during normal limb development.

Salamanders can go through multiple consecutive rounds of regeneration so the authors checked the senescence response over repetitive events of amputation and regeneration. Interestingly, no accumulation of senescent cells was observed after five regeneration cycles over a period of 1.5 years. These results indicate that senescent cells were effectively eliminated during each round of regeneration. Moreover, whereas in other species, including mammals, senescent cells accumulate with age the authors found that senescent cells in heart, spleen and liver did not accumulate in older salamanders. These results suggest that some mechanism of senescent cell clearance functions in both normal and regenerating salamanders. In order to determine whether such mechanism really exists, the authors implanted either senescent cells or normal cells labelled with GFP within newt limbs and followed them over time. Implanted normal cells persisted for at least 40 days and contributed to different structures. However, 80% of the implanted senescent cells were cleared after 2 weeks. These results suggest that salamanders have an active mechanism to get rid of senescent cells. A remarkable observation done by the authors was that when a mixture of 1:1 senescent and normal cells was implanted both cell populations were cleared with time. The reason for that is that these salamander senescent cells were capable to induce senescence in the neighbour cells (a property of the senescence cells seen in other systems). The authors showed here that this effect was mediated by a paracrine factor.

Finally, the authors tried to better characterize this clearance mechanism. Based on previous results on the role of the immune system in both the clearance of senescent cells in other systems as well as in amphibian regeneration, they analysed the role of macrophages in their system. First, they saw that, in vivo, macrophages and senescent cells are in close proximity during limb regeneration. The implantation of senescent cells triggers the recruitment of macrophages to their vicinity, which was not observed when normal cells were implanted. Then, after macrophage depletion, these implanted senescent cells remained over time and were not cleared. Therefore, these results indicate that macrophages were actively involved in the clearance of senescent cells during limb regeneration.

Overall, this study shows for the first time that salamanders possess a senescence surveillance mechanism that operates during regeneration. Remarkably, senescence is strongly induced in the regenerating blastema at mid stages of regeneration. Future experiments should try to determine the biological significance of this senescence upregulation for a successful regeneration.

BMP and FGF signalling pathways cooperate to induce limb formation in urodeles

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.

Sox2 is required for neural stem cell amplification during axolotl spinal cord regeneration

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.

Sustained ERK activation and reprogramming of newt myotubes

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.

Ancient limb-regeneration in tetrapods

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.

Heterogeneity in ependymoglia cells in intact and regenerating newt brain

Even though active neurogenesis in adult mammals is well known and occurs in some regions of our brain, our capacity to replace neurons after an injury or lost is quite limited. In contrast, other vertebrates such as zebrafish and salamanders can regenerate their central nervous system much better. In these animals radial glia like cells (GFAP+) function as neuronal progenitors during regeneration. In the newt brain there are regions (hot spots) in which active neurogenesis is observed during homeostasis in intact animals. However, it is also possible to trigger neuronal regeneration in regions in which neurogenesis is not detected in normal conditions. A recent paper from the laboratory of András Simon (http://www.ncbi.nlm.nih.gov/pubmed/24749074) addresses the heterogeneity of the ependymoglia cells within and outside of the constitutively active niches in the newt telencephalon.

As a first step to isolate neural stem cells (NSCs) the authors tested whether brain cells from different regions were able to form neurospheres in vitro, a typical assay to determine the NSC nature. Neurospheres were indeed formed and included GFAP+ cells that proliferated. Upon media changes cells expressing differentiated neuronal markers were found in those neurospheres indicating that GFPA+ cells have stem cell properties.

In a previous study these authors showed that proliferating ependymoglia cells were mainly localized in hot spots in the newt brain. Here, they addressed the characterization of the heterogeneity of these cells. By analyzing the expression of glutamine synthetase (GS) they could distinguish two different population s of ependymoglia cells. Type 1 GFAP+ cells were positive for GS whereas type 2 GFAP+ cells did not express GS. Type 2 (GS-) cells were found in clusters in hot spots and represent about 32% of the ependymoglia cells. Most of the proliferating ependymoglia cells in hot spots (about 85%) are type 2 cells. The remaining proliferating cells in hot spots (about 15%) are type 1 (GS+). In contrast, in non-hot spots type 2 cells represent only 0,3% of the ependymoglia cells, whereas most of the proliferating cells in these regions are of type 1 (about 90% of the proliferating ependymoglia cells). Thus, type 1 and 2 are found in hot spots but type 2 is practically absent from non-hot spots.

As Notch signaling has a well-known role in the regulation of neural stem and progenitor cells the authors characterized then the expression Notch receptor in these 2 populations of ependymoglia cells in hot spots and non-hot spots. In hot spots their observations are consistent with type 1 cells being GFAP+/GS+/Notch1+ and type 2 GFAP+/GS-/Notch1-. In agreement with this, they found that 94% of proliferating cells in hot spots were Notch1-, whereas most of the proliferating ependymoglia cells in non-hot spots were Notch1+.

Next, as type 2 cells are mainly localized in hot spots the authors hypothesized that they could have a stem cell nature. However, what they found was that a majority of stem cells were in fact type 1 ependymoglia cells. By doing pulse chase experiments with BrdU to detect the long-term label retention characteristic of stem cells together with treatments with AraC (a method to selectively eliminate transit-amplifying cells that divide more frequently than slowly dividing stem cells) the authors concluded that type 1 ependymoglia cells (found in most of the ventricle wall of the telencephalon) have stem cell properties whereas type 2 cells in the hot spots would be transit-amplifying cells.

Then, the authors analyzed how these distinct cell populations responded to the ablation of cholinergic neurons in hot spots and non-hot spots. Upon ablation they found an increase in the proliferation of type 1 and type 2 cells in the hot spots. Remarkably, they also found that in non-hot spots there appeared type 2 cells as well as cells positive for PSA-NCAM, a marker of immature neurons that is not detected in homeostasis in these regions. These last results suggested that neuronal ablation gave rise to the appearance of new neurogenic regions.

Finally, the authors analyzed how interfering with Notch signaling affected the behavior of type 1 and type 2 cells in homeostasis and during regeneration. Upon treatment with the Notch inhibitor DAPT, they concluded that in homeostasis type 1 (stem cell) proliferation is not sensitive to Notch signaling whereas type 2 (transit-amplifying) proliferation is Notch sensitive. During regeneration, type 1 cells in the hot spots are still insensitive to Notch signaling; however, the increase in proliferation of stem cells in non-hot spots is dependent on Notch signaling.

In summary, the authors report here the identification of two distinct subpopulations among the ependymoglia cells in the newt telencephalon: type 1 with stem cell properties and type 2 with transit-amplifying ones. Remarkably, although in the newt telencephalon there are active hot spots, type 1 cells are also found in most of the ventricle wall, which could account for the high neuroregenerative capacity of these animals. In fact, neuronal ablation leads to the appearance of new neurogenic niches in non-hot spots, in which there is an increase in the proliferation of type 1 cells (Notch dependent) as well as the appearance of type 2 and neuronal precursors.

Hedgehog signaling from the notochord during Xenopus tail regeneration

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.

The role of miRNAs in axolotl spinal cord regeneration

miRNAs are small non-coding RNA molecules that play important functions in transcriptional and post-transcriptional regulation of gene expression. miRNAs have been shown to regulate many important biological processes and be linked to several diseases, cancer and neurogenesis, among others. Moreover, miRNAs have been related to liver and heart regeneration. Now a recent paper from the laboratory of Karen Echeverri reports on the function of miR-125b in spinal cord regeneration in axolotls (http://www.ncbi.nlm.nih.gov/pubmed/24719025).

Whereas in mammals the glial scar inhibits axonal regeneration after spinal cord injury, axolotls have no problems in regenerating their spinal cord. Importantly, no glial scar is detected in axolotls after injury. The rationale behind this study was to concentrate on the environment of the injury site in order to analyze whether intrinsic differences could help to explain the differences between the regenerative capacities shown by axolotls and mammals. That is, the authors tried to identify molecules that promote a regeneration-permissive environment in axolotls.

Previous works by the same research group had focused on analyzing the role of miRNAs in axolotl tail regeneration. Here, they carried out a comparative miRNA profiling analysis at different time points after spinal cord injuries in rats and axolotls. The authors chose 1 and 7 days after complete transection of the spinal cord. Of the approximately 4,000 miRNAs of several vertebrate species included in their array, they found 14 showing significant differences after injury in axolotl versus rat. One of the identified miRNAs was miR-125b, previously related to cancer and stem cell differentiation. Before injury, miR-125b expression in axolotl was concentrated mainly in radial glial cells. In rats, also in an uninjured animal, astrocytes had the highest level of expression; however, in this uninjured situation miR-125b showed 8-fold higher expression in axolotls than in rats. One day after amputation the levels of miR-125b in axolotls decreased about 40%, whereas in rats this decrease was less than 1%. The authors hypothesized that this strong and early downregulation of miR-125b in axolotls could be contributing to creating a permissive environment for regeneration. To test it they analyzed how regeneration proceeded after modulating the levels of miR-125b. The overexpression of miR-125b resulted in an aberrant sprouting of the axons on both sides of the injury together with a reduced growth of the axons through the lesion site by 7 days post-injury, when controls showed normal full regeneration. On the other side, the inhibition of miR-125b in axolotls (up to levels similar to those observed in rats) strongly inhibited also axonal regeneration. Interestingly, this inhibition resulted also in a significant deposition of fibrin that reminded the glial scar found in rats. These results suggested that the levels of miR-125b must to be tightly regulated to allow regeneration in axolotls.

Next, they carried out bioinformatics analyses to try to identify putative target genes regulated by miR-125b. Among them, they identified a homologue to Sema4D, a gene that is upregulated in mice at the wound site after spinal cord injury. In uninjured axolotls Sema4D protein was not detected in glial cells, although it was transiently upregulated at 3 days after injury. On the other side, the inhibition of miR-125b resulted in the upregulation of Sema4D at the injury site; conversely, the overexpression of miR-125b decreased the levels of Sema4D. These results suggested that the regulation of Sema4D levels by miR-125b might play an important role during regeneration. In fact, the overexpression of Sema4D at the injury site inhibited axonal regeneration in axolotl. Then, the authors analyzed the relationship between miR-125b, Sema4D and regeneration in rats. To do that, they modulated in vivo the levels of miR-125b after spinal cord injury. By 7 days post-amputation the levels of miR-125b significantly decreased whereas Sema4D levels increased. The overexpression of miR-125b resulted in a reduced expression of Sema4D, further supporting the results obtained in axolotls. Remarkably, these animals showed improved locomotive abilities compared to controls, reduced glial scar formation and increased number of axons projecting into the scar tissue.

Finally, because the overexpression of miR-125b probably affects the expression of many genes other than Sema4D, they performed microarrays analyses to compare control versus treated animals overexpressing miR-125b. Remarkably, they found that many genes involved in glial-scar formation were downregulated, whereas genes related to neurite outgrowth were upregulated after overexpressing miR-125b.

Taking all the results together, the authors propose a model in which miR-125b would promote a regeneration-permissive environment by downregulating the expression of glial-scar genes and other genes such as Sema4D and, at the same time, inducing positive factor for axonal regrowth. The further characterization of this miRNA-regulated pathway in axolotls and rat can uncover fundamental differences between these two species and help us to understand their different regenerative capabilities.

How precise is limb regeneration in axolotls?

Ferchault de Réaumur, in 1712, said that during regeneration “Nature gives back to the animal precisely and only that which it has lost, and she gives back to it all that it has lost”. Since then, we are amazed of seeing that if we amputate, for example, the limb of an axolotl, it regrows up to the same size and shape as the original limb. Moreover, this regenerated limb is as functional as the original one. Similarly, if we cut a planarian in ten pieces, the new ten flatworms that are formed in few days look identical to the original planarian. So, it seems that what Réaumur said 300 years ago, mainly stands true. But, what happens if we analyze the regenerated structures in deep detail? Are they really identical to the original one? In other words, how perfect is regeneration?

Now, a recent paper from the laboratories of Rui Diogo and Elly Tanaka describes the exact pattern of regeneration of 35 muscles that form the arm, forearm and hand of the axolotl’s forelimb as well as a pectoral muscle, the coracoradialis (http://www.ncbi.nlm.nih.gov/pubmed/24692358). This study is part of a larger project to characterize the development, homologies and evolution of many body muscles of all major vertebrate clades. Here, they used transgenic axolotls that expressed GFP in all muscle fibers. They analyzed the pattern of regeneration of these muscles in forelimbs that were experimentally amputated at the level of the arm as well as forelimbs that were amputated “naturally” from bites by other axolotls. In total they analyzed 23 regenerated forelimbs and found out that there were muscle anomalies in 10 of them (43%). This is surprisingly high under the assumption of the precision of regeneration. However, the total number of anomalies in these 23 regenerated forelimbs was only 20, so in average each forelimb had anomalies in only 2,5% of the total number of muscles (n=36) examined. Moreover, the percentage of anomalous muscle was much higher in those regenerates after bite-induced “natural amputations” (3,9% of muscles affected) than in the experimentally amputated regenerates (1,5%). This could be explained by recurrent aggressions affecting different regions compared with the precise and unique experimental amputation.

Another interesting observation was that none of the muscle defects seen in these regenerates was seen in the original limbs analyzed. Remarkably, one of the most common anomalies seen in 35% of the regenerated forelimbs (8 of the 23 regenerated forelimbs analyzed) was the presence of a fleshy coracoradialis at the level of the arm. Usually, this muscle only has a thin tendon at the level of the arm. This anomaly does not necessarily change the main function of this muscle (to flex the forearm). From an evolutionary perspective this anomaly is really interesting because this fleshy configuration resembles very much the fleshy biceps brachii of amniotes, which suggests a parallel between a regeneration defect and a major phenotypic change that occurred during tetrapod limb evolution. In fact, it has been suggested that at least part of the biceps brachii corresponds to the amphibian coracoradialis and, therefore, it could mean that at some point in evolution the tendinous part of the coracoradialis of amphibians had to become associated with fleshy fibers. The authors discuss then that, as suggested by other authors, there is often a parallel between anomalies occurring because of natural or experimental reasons and the normal phenotype found in their closest relatives. This could be interpreted because evolution is highly constrained and, as a consequence, similar phenotypes are often created. In fact, some other of the anomalies in the pattern of the regenerated muscles found in this study are normally found in other non-urodele taxa.

Finally, the authors also investigated the similarities in muscle morphogenesis between regeneration and embryonic development. In both cases, muscles appear to differentiate following a proximodistal and a radioulnar gradient. However, they found here that there is also a ventro-dorsal gradient of differentiation at least for the forearm muscles, that has not been previously seen during embryogenesis, and that would be worthy to further characterize in the future.

In summary, this study addresses how precise is the regeneration of the axolotl limb. On one side they observed that a large percentage of the regenerated limbs showed anomalies in the muscle pattern. But, on the other side, the average number of muscles affected in each limb was really low. Remarkably, they found a common anomaly seen in most of the limbs, which could have further implications for the evolution of muscles in tetrapods.

Regeneration in experimentally induced metamorphic axolotls

Several amphibians are really good in regenerating their limbs after amputation. However, even in these champions of regeneration, this amazing capacity to regrow such a complex structure is often conditioned by the life stage of the animal. Thus, in many cases, especially in frogs, regenerative abilities are very high in tadpoles but decrease significantly after metamorphosis. In fact, this loss of regenerative abilities depending on the life stage is not only observed in amphibians but also in other animal groups, including insects and mammals, in which regeneration power is quite limited after metamorphosis or from post-embryonic stages. Some other amphibians, especially urodeles, retain high regenerative abilities as adults. However, here some variability also exists in terms of the capacity to complete a successful regeneration. In urodeles, another aspect to consider is the influence that metamorphosis may have on their regenerative abilities. Some of them, as some newts, are perfect regenerators as adults but for other species there are unclear results. In any case, it is important to understand how aging, body size and metamorphosis relate to the regenerative capabilities shown not only by urodeles but by other animal groups.

Now a recent paper from the laboratory of James Monaghan and Ashley Seifert has analyzed how inducing metamorphosis in paedomorphic axolotls affect their regenerative abilities (http://onlinelibrary.wiley.com/doi/10.1002/reg2.8/abstract). Axolotls almost never undergo natural metamorphosis but it can be experimentally induced by thyroxine exposure and, therefore, can be a good model to test how body size and metamorphosis affect regeneration. After inducing metamorphosis the authors first checked that the morphology of the limbs of paedomorphic and metamorphic axolotls was quite similar with only slight differences. They used an age-matched cohort of animals with a broad range of body sizes. Contrary to other studies their results suggested that body size (as an independent variable) was not a limited factor for regeneration. Next, they describe how metamorphosis really impairs the regenerative abilities of these axolotls. Although limbs of metamorphic axolotls went through all the normal stages of regeneration, it took to them almost twice as long to regenerate compared to paedomorphs. Moreover, regeneration was not only much slower but also in 100% of the cases the metamorphic axolotls regenerated limbs that showed anatomical defects. These defects included loss of skeletal elements or complete digits or fusion of other skeletal elements. Therefore, metamorphosis reduced regeneration rate and fidelity.

Finally, the authors analyzed cell proliferation dynamics in the blastema of paedomorphic and metamorphic axolotls. By using PCNA, a marker that labels proliferating cells all through the cell cycle, they first showed that there were no differences in the number of proliferating cells. However, when using BrdU to analyze if there were differences in the number of cells in S-phase within those blastemas they found that that metamorphs had a smaller proportion of BrdU-positive cells (relative to total blastema cells), suggesting that there could be a difference in the length of the cell cycle. Remarkably, though, blastemas from metamorphs contained a two-fold higher number of cells.

In summary, this study sows how after inducing metamorphosis in axolotls these animals can still mount a regenerative response after limb amputation. However, regeneration rate and fidelity were severely compromised. Importantly, the defects observed in regeneration after thyroxine treatment were due to metamorphosis and not because of differences in body size or age, which allows to decouple the effects that different parameter may have on the regeneration potential. Moreover, the author suggest that it would be interesting to test whether the putative alterations in the cell cycle timing that they observed after metamorphosis could somehow affect the transcription of some patterning genes required for proper regeneration.

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