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.
In contrast to other vertebrates, mammals cannot regenerate the mechanosensory hair cells in the epithelia of their adult ears after age-related, disease or trauma-induced cell death. Zebrafish can definitely regenerate their hair cells located not only in the inner ear but also within the sensory lateral line. This lateral line consists in rather regularly spaced sensory organs called neuromasts formed by hair and support cells. It is well known that in larval zebrafish hair cells show a strong regenerative ability and, after their ablation, are regenerated from the symmetrical division of the surrounding support cells. Now, a recent paper from the laboratory of David W. Raible reports for the first time on the robust regeneration of these hair cells on aged adult zebrafish and characterizes a slow-dividing subpopulation of support cells that could explain this robustness in hair cells regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25869855).
In this study the authors used several transgenic lines that allowed them to easily visualize and track both hair cells and the surrounding support cells. After neomycin treatment on sexually matured animals, they showed first that 75% of hair cells were ablated by 2 hr and then normal numbers were recovered by 72 hr, a rate of recovery similar to that observed in larval zebrafish. Next, they analyzed whether this regenerative capacity was diminished with age by comparing 1-year and 3-year-old animals. Their results show that 3-year-old zebrafish were still capable of fully regenerating their hair cells after neomycin treatment although it took a little bit longer compared to 1-year-old animals (5 days instead of 3 days). Remarkably, these adult zebrafish were capable of properly regenerating their hair cells after each of 10 sequential rounds of hair cells ablation and regeneration.
In larval zebrafish, regenerated hair cells derive from the symmetrical division of support cells. The authors then checked whether the number of these support cells changed after repeated rounds of regeneration in adults. However, the number of support cells in each neuromasts remained about constant after those experiments suggesting that support cell renewal was tightly regulated during hair cell regeneration. Then the authors combined a transgenic line with labeled hair cells and BrdU staining and found out that the number of BrdU positive hair cells decreased 12 days after BrdU exposure but at the same time the number of hair cells remained quite constant indicating that in normal conditions adult hair cells go through constantly loss and replacement. These results were further corroborated by the use of transgenic lines carrying the photoactivatable fluorescent protein Eos.
Finally the authors tried to understand how support cells are capable of dividing symmetrically to give rise to hair cells during multiple rounds of ablation and regeneration without being depleted themselves. They hypothesized the existence of a subpopulation of slow dividing support cell progenitors. Therefore, they used a transgenic line expressing Eos in all support cells and their rationale was that after multiple rounds of regeneration the red Eos signal present in dividing support cells originating hair cells would be diluted. In contrast, support cells that would not divide or did it much less frequently would retain higher levels of red Eos. Interestingly, they found a population of label-retaining support cells at the anterior end of the neuromasts as well as smaller population with similar characteristics at the posterior end. These results suggested that support cells behave differently depending on their localization within the neuromast. Remarkably, these anterior support cells were much less probable to give rise to hair cells during regeneration.
In summary the authors described here how adult and aged zebrafish are still capable of robustly regenerating the hair cells from their lateral lines. More importantly, they characterized a subpopulation of slow dividing support cells that could originate the hair cells precursors during regeneration and cell turnover. Future experiments should corroborate these findings and further analyze whether the environment around these distinct anterior support cells might have a role as a niche to maintain these support cells in a more quiescent state.
Contrary to mammals, zebrafish can regenerate their hearts after a significant loss of their cardiomyocytes. Taking into account the high incidence of myocardial infarction in humans understanding heart regeneration in zebrafish might provide clues to enhance our limited regenerative abilities. Now, a recent paper from the laboratory of Ken Poss reports on the important role of neuregulin1 (nrg1) t o induce the endogenous heart regeneration program in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25830562).
Nrg1 had been previously identified as having multiple roles in cardiovascular biology in both mammals and zebrafish, although some contradictory reports existed respect to the role of nrg1 on cardiomyocytes proliferation. Therefore, in this paper, the authors addressed the role of nrg1 on the strong regenerative response of the zebrafish heart. Whereas nrg1 expression was rarely detected in uninjured hearts, after the genetic ablation of about 50% of the cardiomyocytes there was a significant upregulation of nrg1 that peaked at 7 days post amputation (dpa), a stage in which cardiomyocytes proliferation also peaks. Double stainings determined that most nrg1 positive cells in the postnatal ventricular wall came from the tcf21+ epicardial derived perivascular cell compartment. Rare overlap of nrg1 and markers of vascular endothelial cells and cardiomyocytes was observed.
Nrg1 signals through the Erbb2 and Erbb4b receptors. The pharmacological inhibition of these receptors decreased cardiomyocytes proliferation during regeneration. On the other hand, gain-of-function experiments showed that overexpressing nrg1 in cardiomyocytes resulted in a strong increase of cardiomyocyte proliferation near the injury site, clearly suggesting an important role of nrg1 signalling on cardiomyocyte proliferation during heart regeneration. Next, the authors sought to determine the effects of overexpressing nrg1 in uninjured hearts. After 7 days of treatment (dpt) they observed a marked increase in cardiomyocyte proliferation especially within the ventricular wall. Over time, this increased proliferation resulted in obvious changes in cardiac anatomy. Thus, the ventricular wall thickened by 76% at 7 dpt, by 265% at 14 dpt and by 459% after 30 dpt. Measurements of cardiomyocyte cell number and size suggested that this enormous thickening of the ventricular wall was mainly due to an increase in cell number. The authors referred to it as nrg1-induced cardiac hyperplasia (iCH). Next, the authors analysed the long-term effects of iCH on uninjured adult hearts. On average after 6 months of treatment those hearts showed a 2.1-fold greater ventricular section area. An increased cardiomyocyte proliferation was still evident at this late stage although the authors also observed some mild fibrin and collagen deposition in some regions of the ventricular wall that were not seen just after 30 dpt.
By measuring physiological parameters and cardiac function the authors did not find any cardiac dysfunction at 3 months of iCH. However, at 8-9 months the animals showed a reduction in swimming endurance, a test based on measuring the ability of the fish to swim against increasing water currents, that is an assay of cardiac function sensitive to cardiac damage or heart failure. Therefore, these results indicated a deleterious effect of nrg1 continuous overexpression over a long period of time.
In a last set of experiments, the authors investigated whether iCH depended on similar mechanisms to those triggered by injury-induced regeneration. During normal development the cortical muscle in the ventricular wall form from a small number of large cardiomyocyte clones with discernable boundaries between them. In contrast, during regeneration there is an increase of cardiomyocyte proliferation at the injury site that generates a mixed conglomeration of small clones in the regenerated ventricular wall. The authors observed that the thickening of the ventricular wall during iCH in uninjured hearts contained mixed small clones something reminiscent of an injury-induced regenerative response. When analysing the molecular signatures of iCH the authors first saw an upregulation of gata4 (an embryonic cardiogenic transcription factor) in the outermost layer of cortical muscle at 7dpt. Previous results had shown that after apex amputation gata4 is upregulated in cardiomyocytes near the injury and is essential for regeneration. Moreover, they observed that after iCH there was a reduction of cmlc2 (cardiomyocyte marker) expression in the cortical muscle and a sarcomere disorganization, which would resemble the dedifferentiation process that occurs during regeneration. Another genes implicated in regeneration, such as tgfb3, fn1 (fibronectin synthesis) and raldh2 (retinoic acid signalling), were also upregulated after iCH. Finally, iCH also induced the formation of a major vascular network throughout the expanded ventricular wall. Overall, all these results suggest that nrg1 is sufficient to induce and maintain a global heart regeneration program involving several cell types.
In summary, the authors report here nrg1 as a potent activator of the heart regeneration program in zebrafish. In the future, it will important to determine how is nrg1 upregulated during regeneration as well as its downstream targets.
An important aspect of stem cell biology is to determine the genes responsible for the maintenance of the different stem cell populations as well as to understand both how they are regulated and how they exert their function. Now, a paper from the laboratories of Ian C. Scott and Bret J. Pearson reports on the identification of the Mediator subunit Med14 as an important regulator of stem cell maintenance in zebrafish and planarians (http://www.ncbi.nlm.nih.gov/pubmed/25772472).
The Mediator complex was first identified in yeasts and its core consists of three modules (head, middle and tail) with an additional kinase module present sometimes. Because in yeast Mediator is located at the promoters of nearly all protein coding genes it has been suggested that it may be part of the general transcription machinery. However, several reports in some plant and animal models do not seem to fully support this model. Interestingly, several subunits of this Mediator complex have been identified as regulators of pluripotency of mouse ESCs. However little is known about the putative role of Mediator on stem cells in vivo.
In this paper the authors characterized the function of the subunit Med14 in stem and progenitor cells and regeneration in two different models: zebrafish and freshwater planarians. First they found that the zebrafish mutant logelei (log) corresponds to mutation of med14. Pleiotropic effects that suggest a developmental arrest characterize the phenotype of this mutant. Accordingly, morpholinos of med14 recapitulate many of these phenotypes, which can be then rescued by the injection of wild-type med14. Analyses of genome-wide transcript levels and mRNA levels did not support a general affectation of transcription in these mutants. In contrast, when the authors focused on stem and progenitor cells and regeneration in the log mutants they observed specific defects. Thus, using different markers they saw that retinal, hematopoietic and gut stem cells were reduced in the log mutant and that med14 seemed to be important in heart progenitor cells. Also, when the tail fin of these animals was amputated it did not regenerate.
In order then to better characterize the putative function of med14 on stem cells and regeneration the authors studied it in planarians, an attractive model in which to study stem cells in vivo. In planarians, as in zebrafish, med14 was ubiquitously expressed including in neoblasts (planarian totipotent stem cells and the only proliferative cells in them). The silencing of med14 by RNAi in intact planarians resulted in a phenotype resembling that obtained after depletion of the neoblasts. That is, the animals curled ventrally, lost their heads and finally lysed. On the other hand, the silencing of med14 also blocked regeneration. After med14 silencing there was a gradual loss of cells expressing the neoblast marker Smedwi-1 (a piwi homologue). There was also a loss of proliferative activity detected with different markers of mitosis and S-phase such as phosphorylated histone H3, histone h2b and pcna. Using a marker of neoblast early progeny the authors found that this cell population was also gradually depleted. In contrast, med14 RNAi did not affect the expression of specific markers for differentiated cell types including neurons, gut cells, muscle, pharynx and eye. Moreover they found a significant increase in apoptosis all throughout the treated animals. These results suggested that med14 silencing affected specifically the neoblasts and their progeny and that transcription in general was not compromised.
In summary, the results presented here indicate that the Mediator complex has an important role in stem cell maintenance in two distant models such as zebrafish and planarians. Further analyses should help determining how the Mediator exerts its function, maybe through establishing the epigenetic landscape essential for pluripotency and stem cell maintenance in these animals.
Regeneration of complex structures is rare within amniotes. A well-known exception is the lizard tail. However, our knowledge on how this process is achieved at the cellular and molecular is very limited when compared to the regeneration of other appendages in another vertebrate groups. Moreover, and contrary to these last appendages the regenerated lizard tail although fully functional is not identical to the original one (for instance, the dorsal root ganglia flanking the central canal of the spinal cord are not regenerated, the scales are smaller and the skeletal muscle regenerates in a different organization). Now the laboratory of MK Vickaryous has revised the current knowledge on lizard tail regeneration, mainly focused on the blastema (http://onlinelibrary.wiley.com/doi/10.1002/reg2.31/abstract).
In many species the tail has a series of fracture planes that allow this appendage to be self-detached or autotomized mainly as defensive strategy. For the majority of lizards these fracture planes are intravertebral; however, many species can regenerate the tail if amputated outside these planes. After amputation a clot is formed distal to the original spinal cord. Just after 24 hours cells begin to proliferate at the wound and form a blastema. At the same time a wound epidermis is formed and proliferates becoming thicker and then referred as apical epithelial cap. As the blastema grows the ependymal tube from the original spinal cord also grows inside the blastema adjacent to the wound epithelium. Differentiation within the blastema appears to occur in a proximal to distal gradient.
Thus, lizard tail regeneration is clearly epimorphic with the formation of a wound epithelium and a blastema. As it happens for the amphibian limb the wound epithelium is essential for regeneration as its removal or prevention of formation inhibits tail regeneration. However, very little is known about the molecular interactions between the wound epithelium and the blastema. Concerning the blastema its cellular origin is mainly unknown. In light of what is known from the blastema of other appendages in amphibians and zebrafish, it seems probable that the lizard tail’s blastema will be formed also by lineage-restricted cells, although experimental confirmation is needed. After amputation several cell populations begin to proliferate within the blastema. As differentiation occurs, proliferation decreases although chondroblasts and myoblasts continue to proliferate until the tail is fully regenerated. The presence of resident/stem progenitor populations has been suggested for the skeletal muscle and ependymal tube, although their existence needs to be unambiguously confirmed.
During tail regeneration the ependymal tube seems to have a central role in the reestablishment of the primary axis. Thus, it organizes and guides unmyelinated tracts from the original tail and likely induces and directs the outgrowth of the new tail. In fact, the ependymal tube is one of the first structures that appear within the blastema (at 4 days). Importantly, if the spinal cord adjacent to the wound is ablated or blocked from outgrowing, regeneration is inhibited. What seems to be important here are the ependymal cells that line the central canal of the spinal cord. More amazingly, transplants of ependymal tube to ectopic locations can stimulate regeneration. Finally, the ependymal tube has been also suggested to have a central role on the patterning of the regenerated tail and the induction of cartilage.
Although the conserved signaling pathways that act during tail regeneration remain mainly uncharacterized it has been reported that many blastema cells are positive for phosphorylated SMAD2, a mediator of the TGFB/activin pathway, and the epithelial-mesenchymal transition markers Snail1 and Snail2.
In summary, many questions need to be answered concerning lizard tail regeneration especially on the source (stem/progenitor cells/dedifferentiation/transdifferentiation) and properties of the blastema cells (proliferative or non-proliferative).
Many animals are able to regenerate different types of appendages (understood as body wall outgrowths). Among vertebrates, limbs and tails from salamanders and lizards are well-known examples. On the other side, some invertebrates also regenerate appendages such us parapodia, tentacles, opercula, palps and gills. Molluscs comprise a very diverse phylum with an extreme morphological diversity. Up to date, no known representative has been shown to be able to regenerate the whole body. However, several mollusc species can regenerate a variety of structures such as: foot, tentacles, siphon, shell and mantle, and even the head. Cephalopod molluscs are well known for their capacity to regenerate their arms. However, very little quantitative data is available about this process and even less about the cellular and molecular mechanisms involved. In a paper from the laboratory of Jedediah Tressler Nathan J. Tublitz the authors provide a detailed description and quantification of the regeneration process in two species of cuttlefish (http://www.ncbi.nlm.nih.gov/pubmed/23982859). Studying arm regeneration in cephalopods is also important as these structures are as complex as many vertebrates appendages.
It is also obvious that an initial detailed description and quantitative data on the regeneration process in terms of growth rate, behavioural changes, timing of regeneration and functional recovery is necessary prior to tackle the cellular and molecular mechanisms that regulate it, especially in those non-model animals.
First, the authors present the data on Sepia officinalis in which the right 3rd arm was amputated from nine juveniles. In all cases, the amputated arms were regenerated by 39 days, after which the newly formed arms were indistinguishable from the contralateral control arms. They divided the regeneration process in 5 stages: at stage I (days 0-3) the leading edge of the arm appeared smooth with little bleeding. Two days after the amputation the regenerating arm appeared frayed and covered with a mucus-like substance. At this stage only a few new suction cups and no new chromatophores were observed. As a consequence of the amputation the behaviour of the animals was significantly altered as they showed: unbalanced swimming, impaired prey manipulation for ingestion, altered normal body posturing behaviours and lack of any colour change behaviour. Stage II (days 4-15) was characterized by the smooth, slightly hemispherical appearance displayed by the leading edge of the regenerating arm. Growth across the entire width of the arm was symmetrical. New suction cups and chromatophores were seen at this stage. Also, a normal balanced swimming was seen and from day 9 normal food manipulation reappeared. Stage III (days 16-20) began with the appearance of a growth bud on the lateral side of the leading edge, resulting in an asymmetric shape of the regenerating arm. The new arm kept growing at a higher rate that the contralateral control and new suction cups and chromatophores were added at the distal tip. By the end of this stage the regenerating arm was also used for normal body postures. Stage IV (days 21-24) was defined by the emergence of an elongated tip from the growth bud. Suction cup regeneration appeared to be completed by the end of this stage. In the final stage V (days 25-39) the elongated tip took on a tapered appearance. New chromatophores were added until their density in the tip resembled that from control arms. Here, all the checked behaviours, including the brown tip behaviour, were recovered.
In the other species, Sepia pharaonis, arms were also regenerated in 39 days and followed the same 5 stages as in S. officinalis. Based on the location of the new suction cups, chromatophores and the presence of the growth bud and elongated tip, it seemed that new tissue was added directly to the tip of the regenerating arm. Whereas the growth rate of the regenerating arm was quite constant throughout regeneration in S. pharaonis, it varied depending on the stage in S. officinalis.
In summary, in this paper the authors report on the fine description of arm regeneration in two species of cuttlefish at the morphological and behavioural recovery levels. Next step should be to get insights into the cellular and molecular processes governing this regenerative process.
One of the on-going debates in the regeneration field concerns how the regenerative capabilities shown by different animal groups have evolved. When considering the animal phylogeny we can see how most phyla contain species capable of regenerating. However, a huge variability exists in: 1) the regeneration power shown by closely related species and 2) the biological level of regeneration as depending on the model they can regenerate only specific cell types, or some tissues and/or organs, or structures and complex parts (for example, a limb) or, finally, the real champions capable of regenerating the whole body.
A recent paper by Alexandra Bely and colleagues discusses about the evolution of regeneration, especially within the spiralians (http://www.ijdb.ehu.es/web/paper/140142ab/regeneration-in-spiralians-evolutionary-patterns-and-developmental-processes). As the authors raise, an important question is whether regeneration variation among bilaterians is the results of regeneration losses, independent gains or a combination of both. That is, was regeneration a feature that was already present in the last common ancestor of all bilaterians and has been lost in some taxonomic groups? or, alternatively, is something that has independently appeared in some groups and not others?
In order to address these questions it is absolutely necessary to gather as much information as possible about the regeneration capabilities of as many animal groups as possible and, more importantly, characterize the cellular and molecular processes that guide regeneration in those animals. Also, it is important to understand why very closely related species can differ significantly in their regenerative capabilities. In this review, the authors focussed on the Spiralia, a large and diverse protostome clade composed of 13 phyla including annelids, molluscs, nemerteans, platyhelminthes and rotifers. Importantly, there is an important variability of regeneration not only between different spiralian phyla but also within them.
Thus, annelids include species that can regenerate every part of the body, including some that can regenerate a whole animal from a single body segment, as well as species totally incapable of regenerating a single segment lost. In general, the capacity to regenerate posterior segments is very broadly distributed within the phylum. In contrast, the ability to regenerate anterior segments is much more variable and, in fact, the failure to regenerate anterior segments has been shown in over a third of the families from which data are available. Nemerteans can undergo growth and degrowth indicating processes of remodelling. Some animals can be maintained starved for over a year shrinking in size but otherwise apparently happy. Among them, regeneration of the proboscis (used for prey capture), tail and head occurs in some groups, and some species can regenerate the whole animal from a tiny body piece. Posterior regeneration is not in general very well documented because the lack of easily scorable structures. Anterior regeneration appears to be very limited within this group although some species of one particular family can regenerate a complete head. However, this seems to be an exception within this phylum, which could imply that it might be a regeneration gain of this particular family.
Among Platyhelminthes, the triclads (planarians) are the best known in terms of their regenerative capabilities. However, it is also true that a number of groups of this phylum have much more limited regenerative capacities. Also, within this phylum posterior regeneration appears to be more widespread than anterior regeneration. Finally, within molluscs we do not have any representative capable of regenerating the whole body. However, different species can regenerate specific structures such as the foot, anterior neural elements, tentacles and even the entire head in some gastropods.
Next, the authors review what is known about the cellular and molecular basis of regeneration within these different phyla. Thus, in annelids after amputation there is a rapid muscle contraction to seal the wound. During the very first stages of wound healing and regeneration, proliferation throughout most of the body seems to be shut down. At the same time there is a large cell migration response towards the wound. After wound healing, cells near the wound start proliferating forming a regenerative blastema. The origin of the regenerative cells within the blastema seems to come from the proliferation of the three tissue layers close to the wound. The role of annelid neoblasts (undifferentiated cells) in regeneration is still under debate. Also, several genes have been shown to be expressed within the blastema including markers of stem cells and germline as well as Hox genes. Interestingly, all these genes expressed within the blastema are also detected during normal growth in the posterior growth zone. This suggests a shared molecular mechanism between regeneration and growth. Finally, regeneration does not uniquely imply the formation of new tissues and structures but also a remodelling of the pre-existing tissues.
In nemerteans, amputation is followed by muscle contraction and wound healing followed by a phase of cell proliferation and the formation of a regenerative blastema, much more evident during anterior regeneration than in posterior regenerates. Unfortunately, the origin of the regenerative cells of the blastema is obscure, and although some old studies pointed to the role of some putative undifferentiated and totipotent cells scattered in the extracellular matrix, more recent studies does not seem to support the existence of such undifferentiated cells. Also, very little is known about the genetic program triggered during regeneration within the blastema cells, except some studies reporting the expression of pax6 and otx in the regenerating central nervous system. Within platyhelminthes, most of the cellular and molecular data of the regenerative process comes from planarians, macrostomids are providing also some interesting data. In planarians, wound healing is followed by the local proliferation of totipotent stem cells (known as neoblasts) closed to the wound that originate a regenerative blastema in which the new structures differentiate. Remodelling of the pre-existing tissues is also necessary to achieve normal body proportions of the regenerated animal. Recently, many papers have reported on different genes and signalling pathways that regulate proper regeneration in planarians. However, much more data should be provided from those taxonomic groups that have either poor regenerative capabilities or for which the cellular and molecular basis of their regenerative capacities are currently unknown. Finally, very little is known about the cellular and molecular processes involved in regeneration in molluscs. A recent report on octopus arm regeneration suggests that a mass of mesenchymal undifferentiated cells would accumulate below the wound forming a highly proliferative blastema.
From all these data and comparative analysis in these spiralian phyla the authors draw four main conclusions: 1) the ability to regenerate the whole body seems to be present in only a subset of representatives of each of these groups. From a phylogenetic perspective numerous increases and/or decreases in regeneration ability have occurred across these phyla. This raises that the possibility that regeneration may not be homologous across them needs to be considered; 2) posterior regeneration appears to be more widespread than anterior regeneration; 3) all phyla include a blastema stage, although the origin of the regenerative cells that form it may be different, and 4) in all these phyla the capacity for continuous growth and degrowth is well documented, suggesting a mechanistic relationship or common set of elements and features between these processes and regeneration.
In summary, how the capacity of regeneration has evolved is also a fascinating field of study that requires much more sampling and data collection for the required comparative analyses.
During zebrafish fin regeneration different genes and signalling pathways are locally upregulated in the wound area and play important roles to trigger a successful regenerative response. However, less is known about the existence and function that putative factors provided by tissues away from the wound could have during these regeneration events. A recent paper from the laboratory of Atsushi Kawakami reports on a diffusible signal derived from hematopoietic cells that would support cell survival and proliferation during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25533245).
The authors studied a mutant called cloche (clo) previously identified from a phenotype that lacks most hematopoietic and endothelial cells. In wild-types (fish larvae), the fin fold was normally regenerated (in size and shape) by 3 days after amputation. In contrast, clo mutants displayed aberrant regeneration with the formation of tissues with a blister-like appearance. The authors thought that this might represent dying cells so they checked with TUNEL assay to find out a large number of dying cells in this region, compared to wild-types. Uncut clo mutants did not display higher levels of cell death so it seemed that this increase in cell death in clo mutants was regeneration-dependent. This was further confirmed because in a wound healing assay there was no increase in cell death in these mutants. This increase in cell death was accompanied by a decrease in cell proliferation as assayed by BrdU labelling. Again, here, the cell proliferation levels in the head and trunk (away from the wound) were normal compared to wild-types.
Immediately after amputation (0,5 hours), necrotic cell death was similarly induced in the injured region of wild-types and clo mutants. This necrotic cell death disappeared by 6 hours in wild-types and clo mutants, but then at 12 hours after amputation clo mutants displayed a high number of TUNEL positive cells. This cell death was mostly suppressed when overexpressing Bcl2 (a negative regulator of apoptosis) in these mutants, suggesting that cell death was most probably caused by a Bcl2-regulated apoptosis pathway.
In clo mutants this increased apoptosis coincided with the stage in which regenerative cell proliferation begins. The authors checked then the expression of junb and junbl early markers induced in the wound epithelium and the blastema, respectively. These two genes appeared to be normally induced in the clo mutants, suggesting that the initial regenerative response might be normal in them. Next, they carried out some transplantation experiments between clo mutants and wild-types to investigate the cell autonomy of Clo protein function during regeneration, concluding that Clo function was non-cell autonomous for the survival of the regenerative cells. These results prompted them to wonder whether the lack of hematopoietic cells in clo mutants was responsible for these reduced cell survival during regeneration. Thus, they analysed tal1, another hematopoietic mutant, and found out similar phenotypes: impaired fin fold regeneration with increased apoptosis and reduced cell proliferation. As the hematopoietic lineage contains several cell types they wanted to identify the cell types required for the survival of the regenerative cells in these mutants. In red blood and endothelial cell mutants they did not observed apoptosis of the regenerative cells. However, when they knocked down the myeloid lineage through the silencing of pu.1, a transcription factor required for myeloid cell differentiation, they obtained similar results to those observed in clo and tal1 mutants, suggesting that myeloid cells would have an important function on the survival of the regenerative cells.
Finally, they wanted to investigate how this putative signal was mediated from the hematopoietic tissues to the amputation site. They developed an in vitro assay for a tail explant cultured for 12 hours post-amputation. Wild-type and clo and tal1 mutant explants normally induced the expression of junb and junbl. Whereas in wild-types no apoptosis was observed, explants from clo and tal1 mutants displayed an induced cell death. These results suggest that the defects observed in clo mutants could not be caused by an impaired circulation. Remarkably, when the mutant explants were treated with body extracts prepared from wild-types there was a rescue of the apoptosis. On the other hand, body extracts from the mutants did not rescue the apoptosis in the tail explants from those mutants. These results suggested that the survival of the regenerative cells might be supported by a diffusible factor in the wild-type body.
In summary, this study suggests that a diffusible, and still unknown, factor derived from the hematopoietic cells would support the survival and proliferation of primed regenerative cells during fin fold regeneration in zebrafish.
Mammals cannot regenerate their hearts despite that some studies have reported that newborn mice can regenerate the heart apex if it is amputated during their first days of life. Also, in some special pathological or physiological conditions adult cardiomyocytes can be forced to re-enter the cell cycle and/ or dedifferentiate. However, these responses cannot sustain the proper regeneration that should take place after, for example, an acute myocardial infarction. In contrast, zebrafish cardiomyocytes retain their ability to dedifferentiate and re-enter the cell cycle throughout their lives, which explain the amazing capacity of these animals to regenerate a large portion of their hearts. Experimentally, regeneration studies in the zebrafish heart are carried out after either amputation or cryoinjury.
Now a recent paper from the laboratory of Anna Jazwinska reports on the spatial and temporal dynamics of cell proliferation and differentiation during cryoinjury-induced heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25557620). The authors first used MCM5 as a marker of the G1/S phase of the cell cycle and phospho histone H3 (PH3) to label mitotic cells in G2/M. Moreover, they used these markers on transgenic zebrafish expressing GFP under the control of the cardiac specific promoter cmlc2. During regeneration the initial fibrotic tissue was progressively replaced with new myocardium, which resulted in the decrease of the infarcted area relative to the entire ventricle. As expected, regeneration was characterized by an increase in cardiomyocytes (CM) proliferation, quite evident by 7 dpci (days post-cryoinjury). During the progression phase at 17 dpci the number of proliferating cells declined and at 30 dpci (termination phase) the mitotic levels had declined up to the levels in uninjured fish. However, at this late stage, the number of MCM5-positive CM still remained significantly higher compared to uninjured animals.
Then, the authors determined the spatial distribution of the dividing CMs relative to the injury border. During the initiation phase (7 dpci) there was a graded distribution of PH3 mitotic cells. Thus, 50% of the PH3 cells were within the first 100 mm adjacent to the cryoinjury border; 20% of them were found between 100 and 200 mm from the border, and the remaining 30% were distributed from the 200 to 1000 mm. Thus, most of the mitotic cells were located at the close vicinity of the wounded area. This graded distribution of mitotic cells was not detected during the progression phase (at 17 dpci) when the PH3 cells were nearly evenly spaced within the entire myocardium. It is important to point out that at this later stage the number of PH3 cells was still significantly higher compared to an uninjured heart. Next the authors performed some BrdU labelling and found out, as expected, that the majority (65%) of CMs located in the regenerated myocardium were BrdU-positive, indicating that they derive from newly generated cells. In the adjacent region (0-100 mm from the regenerated myocardium) about 30% of the CMS were BrdU-positive. Beyond this zone and in all the subregions that the authors checked they found an even percentage of BrdU-positive CMs (about 10%), that again was higher compared to uninjured hearts. Overall, these results suggest that in addition to a local rapid and strong proliferative response to cryoinjury there was also a systemic response all throughout the pre-existing heart.
Finally, the authors analysed the formation of immature CMS during regeneration. To do that they took advantage of an antibody raised against a mammalian neonatal muscle and that binds the N-terminal end of slow b/cardiac myosin heavy chain. In zebrafish this antibody seemed to label embryonic undifferentiated CMs. During regeneration (at 7-10 dpci) the authors found a significant number of CMs positive for this antibody at the injury border within a distance up to 100 mm from the post-infarcted tissue. No labelled cells were found in regions beyond this zone. During the progression stage (17 dpci) these positive CMs lost their initial homogenous alignment along the injury border and intermingled with the regenerated mature cardiac tissue. At 30 dpci almost no labelling was detected. Altogether, the authors concluded that during the initial phase of regeneration, undifferentiated CMS appear at the leading edge of the dedifferentiating adult myocardium and would provide a source of new CMs during regeneration.
In summary, the authors propose that upon cryoinjury a local epimorphic regenerative response is triggered within the 100-200 mm adjacent to the injury border and in which a significant increase in immature and proliferating CMs was detected. This initial response in this blastema-like region would be further reinforced by a systemic compensatory regenerative response within the entire pre-existing myocardium.
What are the exact mechanisms that control the re-establishment of axial polarity remains as an important question in many regeneration models. In freshwater planarians several studies in the last years have identified the Wnt/β-catenin signalling pathway as pivotal for the establishment and maintenance of the anteroposterior (AP) axis. Thus, several Wnts and Wnt receptors are expressed in the posterior regions whereas different inhibitors of Wnt signalling are expressed at the anterior end. Consequently, the inhibition of this pathway by silencing either β-catenin or some Wnt genes (such as Wnt1) results in the regeneration of heads instead of tails. Even in intact animals the silencing of these genes transforms the tail region into a head with a proper brain. Conversely, the activation of the Wnt/β-catenin pathway in anterior regions leads to the transformation of those heads into tails. Therefore, it seems quite clear that Wnt/β-catenin is required to specify posterior fates in these animals.
Now, a paper from the laboratory of Kerstin Bartscherer has aimed to identify genes regulated by this pathway (http://www.ncbi.nlm.nih.gov/pubmed/25558068) that could be involved in controlling tissue polarity. In order to identify target genes the authors carried out RNAseq experiments after silencing Smed-β-catenin1 in intact animals. Initially they identified 440 downregulated and 348 upregulated transcripts. Among the downregulated genes they found previously described posterior genes and, conversely, upregulated genes included some known anterior genes. Then, they selected 70 genes downregulated after β-catenin silencing and analysed their expression patterns by in situ hybridization. Of them, 35% displayed differential expression along the AP axis with high expression in the tail. Among the other genes, 21 of them were expressed within or around the digestive system. When they analysed the expression of most of these genes after β-catenin RNAi, they found that their expression was strongly inhibited, validating thus the RNAseq data. Also, they found that many of the upregulated genes after β-catenin RNAi were mainly expressed in anterior regions, as expected.
Next, they selected some of these genes that showed a graded expression along the AP axis and analysed in more detail their expression by FISH. These experiments clearly showed this graded expression, high in the tail and lower towards more anterior regions, strengthening the hypothesis that a β-catenin activity gradient may control gene expression. Recently, it has been described that collagen-positive subepidermal muscle cells express several position control genes (PCGs), which has lead to propose that these muscle cells could somehow provide positional information along the AP axis. Here, the authors show that some of the putative novel targets of β-catenin are also expressed in those posterior muscle cells. In addition, some of these genes were expressed in intestinal muscle cells, which suggests that the gut musculature could be also a source of PCGs. Then, and in order to determine whether planarian neoblasts (totipotent stem cells) differentially expressed the candidate genes sp5 and abdBa along the AP axis they isolated different FACS-sorted cell populations from anterior and posterior regions and quantified their expression in those cell fractions. Interestingly, they found that sp5 and abdBa transcripts were higher in the stem cells from posterior regions compared to anterior neoblasts, suggesting that planarian neoblasts may respond to graded β-catenin activity along the AP axis.
Notum is a secreted Wnt inhibitor that, in planarians, is expressed at the anterior tip of the animal and whose silencing leads to the regeneration of heads instead of tails. It has been proposed that notum regulates early polarity decisions through the inhibition of β-catenin activity at anterior-facing wounds. As the authors reasoned that notum inhibition might induce β-catenin-dependent genes they carried out RNAseq experiments after notum RNAi at 18 hours of regeneration, a stage in which polarity decisions are supposed to occur. By comparing the two sets of genes from the two different RNAseq experiments, they found that 38 of the downregulated genes after β-catenin RNAi were induced after notum silencing. Conversely, 16 of the upregulated genes after β-catenin RNAi were inhibited after notum RNAi. Thus, these 54 genes could represent the genes involved in early polarity decisions during planarian regeneration. Remarkably, 33 out of these 54 genes were also present in a previously generated set of Wnt/β-catenin target genes in zebrafish, which suggests that the common requirement of the Wnt/β-catenin pathway during regeneration in planarians and zebrafish may include a shared set of target genes.
One of the novel candidate target genes identified here was teashirt, a Tsh-related zinc finger protein, a gene that appears to act as modulator of Wnt signalling during Drosophila and Xenopus development. In planarians, teashirt (tsh) was expressed all throughout the central nervous system as well as in a graded pattern in the mesenchyme with highest expression in the tail. Tsh expression was induced upon amputation and, as predicted, it was inhibited after β-catenin RNAi and induced after β-catenin overactivation. Remarkably, a tsh homolog in zebrafish was detected in fin regenerates at 3 days after amputation, being this expression also dependent on the Wnt/β-catenin pathway further supporting an evolutionarily conserved role of Wnt/β-catenin in ensuring correct tissue identity and/or patterning during regeneration. Going back to planarians, the silencing of tsh resulted in two-headed planarians, a phenotype reminiscent of β-catenin and Wnt1 RNAi. These results indicated that tsh would be required to suppress anterior fate at a posterior-facing wound. During posterior regeneration Wnt1 is rapidly induced in the stump. Tsh co-localized with wnt1-positive cells both in intact and regenerating tails. Morevover, tsh was also expressed in neoblasts and that expression seemed to be higher in neoblasts from posterior regions.
In summary, the authors show here that tsh is expressed in wnt-positive cells, probably subepidermal muscle cells, as well as in a subpopulation of neoblasts in both cases in a β-catenin-dependent manner. Tsh could be then a downstream transducer of Wnt signalling important to regulate AP polarity.