Anterior regeneration in the annelid Cirratulus cirratus

Among invertebrates some annelid species show also remarkable regenerative abilities. Within this group of animals, however, it is not rare to find species that can regenerate posterior regions but not anteriorly. Also, regeneration has not been extensively studied in all taxa. Now, a recent paper from the laboratory of Michael Weidhase and Conrad Helm (http://www.ncbi.nlm.nih.gov/pubmed/25392962) describes for the first time the process of anterior regeneration in the annelid Cirratulus cirratus, focussing mainly in the musculature and central nervous system (CNS). The authors used phalloidin to label the muscle and antibodies against serotonin and FMRFamide to observe the regenerating CNS.

As for the body wall musculature, it is mainly composed of an outer layer of circular fibers and an inner longitudinal muscle layer. The dorsal longitudinal muscles form a plate that covers the whole dorsal body. Ventrally, there is a medial and two ventro-lateral strands of longitudinal muscle. On the other hand, the CNS is a rope-ladder-like system. The ventral nerve cord consists of two main strands and each of them exhibits one ganglion per body segment. From these ganglia there are three major nerves that extend laterally. At the first chaetigerous segment both strands of the ventral nerve cord separate from each other to form the circumesophageal connective surrounding the mouth opening. This connective splits into a dorsal and a ventral root. The brain is located at the anterior end of the circumesophageal connective. Anti-serotonin immunostaining revealed two brain commissures: a stronger stained anterior commissure connected to the ventral root of the circumesophageal connective and a slighter stained posterior commissure connected with the dorsal root.

After amputation, the wound was closed and a blastema was formed within the first week. Then this blastema elongated anteriorly and about 10 days after decapitation the new mouth opening was evident. By the end of the second week the first outgrowing tentacles appeared. The whole regeneration process took place in about 1 month, although the regenerated head showed some differences with the original respect to the number and length of tentacles and branchiae.

By day 6 of regeneration a blastema was formed and contained outgrowths from the pre-existing longitudinal muscle fibers. From that time the longitudinal musculature kept growing and organized into muscular strands. By day 10 the ventral longitudinal strands reached their most anterior position, surrounding the regenerated musculature of the new mouth opening. At this stage the first circular muscle fibers appeared.

Concerning the nervous system, by day 6 after decapitation, a structure represented by three nerve loops was visible inside the blastema, one median loop and two laterals. At this stage the median loop was folded backwards and was connected to the inner bundles of both strands of the ventral nerve cord. On the other hand, the lateral loops were already oriented anteriorly and were connected to the outer bundles of the ventral nerve cord. At day 8 of regeneration the median loop was also oriented anteriorly. This tripartite loop-like structure has not been described in other regenerating annelids. Whereas the authors favour that the median loop will originate the ventral root of the regenerated circumesophageal connective (visible by day 12), they did not obtain clear data about the transition from the lateral loops to the dorsal root. Finally, the brain commissures were visible by day 14 of regeneration. At this stage, the regenerated nervous system elongated together with the blastema; however, distinct ganglia and lateral processes in the new nerve cords were still missing by day 18. Later, the first signs of body segmentation and differentiation of distinct ganglia and lateral nerves were observed.

In summary, this paper describes for the first time anterior regeneration in the annelid Cirratulus cirratus. Further studies are necessary to describe in more detail this process as well as to determine the origin of the regenerative cells that form the blastema and the new muscle fibers and central nervous system, among all the other anterior tissues.

Schwann cells and DCC guide regenerating axons towards their original path

In contrast to the central nervous system the axons of peripheral nerves are able to regenerate in many vertebrates, including mammals. How this regeneration is achieved and the cellular processes involved have been described in many studies. An important point here is that the cellular environment plays an important role in promoting this axonal regrowth. Among these elements, Schwann cells have been described as required for peripheral axons regeneration. Now, a recent paper from the laboratory of Michael Granato reports on the observation in vivo of axonal regeneration in zebrafish as well as the requirement of Schwann cells to direct those axons towards their original paths (http://www.ncbi.nlm.nih.gov/pubmed/25355219).

Here, the authors use transgenic zebrafish expressing GFP in motor neurons and RFP in Schwann cells to follow in vivo the regeneration of fully transected motor nerves in larvae at 5dpf (days post fertilization). The first thing they describe is how upon transection the amputated distal axons were degraded and fragmented leaving behind axonal debris and denervated Schwann cells. Then, Schwann cells suffered dramatic changes from a tube-like morphology to a shorter and more rounded one. As motor axons regrew into their original paths, Schwann cells membranes also reverted to their normal thinner appearance.

In order to define in vivo the role of Schwann cells the authors used different mutant strains that lack differentiated Schwann cells (mutants for sox10, erbb2, erbb3 and nrg1 type III). In all these mutants, motor axons developed normally through 5 dpf. However, upon transection, axon regeneration was severely affected in all mutants lacking Schwann cells. In them, in 50-80% of the transected nerves the regenerated axons failed to target their original paths and instead ectopically grew into more lateral territories. These results suggest that Schwann cells directed regenerating axons. One possibility could be that proper axonal regeneration would depend on some factor produced by Schwann cells during development. To address this question the authors used a transgenic line in which Schwann cells develop normally but can be eliminated at the requested time point of development. Then, the authors killed the Schwann cells at 3,5 dpf and transected the motor nerves at day 5 dpf. In this context, the regenerated axons also failed to find their original targets as previously described for the sox10 mutants, suggesting that Schwann cells must be present during regeneration to direct re-growing axons. In another set of experiments the authors show how in sox10 mutants the regenerative growth cones initially sprouted and extended from the proximal stump in multiple directions as it happens in wild-type larvae. However, and in contrast to wild-type, axons continued to extend in all directions without going back to their original paths.

Distal denervated Schwann cells could exert this guiding role either by providing a physical substrate for the regenerating axons or by producing some factors that would direct the re-growing axons towards their original paths. To try to distinguish between these two possibilities the authors used a transgenic line in which the degeneration of the amputated distal nerves was delayed by more than 1 week. They crossed this line with the sox10 mutant line so an axonal scaffold distal to the transection gap completely lacking Schwann cells was formed. In mutants for axonal degeneration but with normal Schwann cells, motor axons regenerated normally indicating that those distal amputated axons had no inhibitory action on regeneration. However, in the double mutant with sox10, the regenerating axons failed to find their original paths. In other experiments, the authors transected only half of the axons in the motor nerve to create a continuous axonal scaffold devoid of Schwann cells (in sox10 mutants). There, the presence of this axonal scaffold could only partially compensate for the role of Schwann cells as 50% of the transected nerves showed aberrant abnormal projections compared to the 90% observed in fully transected nerves lacking Schwann cells. Overall, these results suggest that Schwann cells provide more than just a permissive substrate for regeneration.

Finally, the authors analysed the role of dcc (deleted in colorectal cancer) a receptor for netrin, a well-known attractive guidance cue for axonal growth. At 5 dpf dcc was expressed in motor neurons as well as in Schwann cells, whereas netrin1b was expressed in Schwann cells before and after transection. Also, dcc was expressed in motor neurons during the first stages of regeneration. Then the authors used a mutant line for dcc in which, interestingly, motor axons and Schwann cells develop normally. However, upon transection, about 40% of the nerves extended axons not only to their original paths but also ectopically into more lateral territories.

In summary, the authors show here the process of motor axon regeneration in vivo in zebrafish. Schwann cells are required for guiding the regenerated axons to their original paths and do it probably not only by providing some physical substrate but actively producing some factor. Future experiments should determine if netrin could be one of such factors.

Planarian regeneration depends on specialized neoblasts

A long-standing question within the field of planarian regeneration relates to the nature of the neoblasts, planarian totipotent stem cells. These cells are the only cells with proliferative capabilities in these animals and are absolutely indispensable for regeneration. Based on morphological criteria (size, shape, nucleus/cytoplasm ratio) about 20-30% of the planarian cells have been considered as neoblasts. However, it has not been clear for many years whether all these cells represent a uniform neoblast population with identical o similar proliferative abilities and potentiality or, alternatively, neoblast comprise a quite heterogeneous cell population respect to these criteria. Recent studies from several laboratories have identified several genes and transcription factors that are expressed in different populations of neoblasts and are required for the differentiation of specific cell types. Based on these data Peter Reddien proposed two models for planarian neoblasts: 1) the naïve model in which neoblasts are a rather homogeneous cell population with the same potentiality and in which fate specification occurs only in the non-dividing neoblast progeny; in contrast, 2) the specialized model predicts that neoblasts are an heterogeneous population containing many different lineage-committed dividing cells.

Based on several recent studies planarian neoblasts seem to follow the specialized model as several cell type-specific genes are co-expressed with Smedwi-1, a planarian piwi homologue, considered as a neoblast marker. Now, a recent study from the laboratory of Peter Reddien further supports the specialized neoblast model (http://www.ncbi.nlm.nih.gov/pubmed/25254346). In this work, the authors first purified by FACS neoblast that were in the S or G2/M phases of the cell cycle (dividing neoblasts) and analysed their expression profiles by RNAseq. Then, they focussed on transcription factors as these proteins regulate cell differentiation. Here, they identified a list of transcription factors that were upregulated in dividing neoblasts during regeneration. To validate these results they next carried out in situ hybridizations on purified dividing neoblasts and all of the transcription factors tested were expressed within these cells although, evidently, with different percentages of positive cells.

They extended then these analyses to other previously characterized transcription factors specific for different cell types. Again, here, they identified a percentage of dividing neoblasts in which these factors were also expressed. Next, they focussed on the planarian nervous system as a target to test the specialized neoblast model as many different neuronal subpopulations have been identified. From the analyses of the transcription factors identified in their RNAseq experiments as well as for the search of conserved factors with conserved functions on the development of the nervous system they found 26 neural transcription factors that were expressed in dividing neoblasts. One of these factors, a klf homologue was coexpressed with cintillo in mechanosensory cells located around the head periphery. Remarkably, klf RNAi lead to the absence of these sensory cells during regeneration, despite these treated animals were capable to normally differentiate many other neuronal cell types. Similarly, a pax3/7 homologue was found to be expressed in the medial region of the brain in cells some of which also expressed a dopamine β-hydroxylase (DBH) gene. Animals in which pax3/7 was silenced by RNAi regenerated a significant reduced number of DBH positive cells. Altogether, these results suggest that these transcription factors would be necessary for the differentiation of specific neural lineages from distinct progenitor neoblast subpopulations.

Importantly, the authors also found that transcription factors associated to specific cell lineages (pharynx, central nervous system, eye, protonephridia or muscle) were not co-expressed in the same dividing neoblasts. These strongly suggest that distinct neoblast populations expressed specific combinations of transcription factors associated to different differentiated cell types.

In summary, this study reports 36 transcription factors expressed in dividing neoblasts from regenerating planarians. These factors are expressed in different cell types and tissues in adult planarians, which suggests that they may specify distinct subpopulations of lineage-committed dividing neoblasts, further supporting the specialized model.

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.

Enteric nervous regeneration in sea cucumbers

Sea cucumbers are well known for their ability to regenerate their digestive system as they can eviscerate and then regenerate their internal organs, including the gut. After evisceration the remaining mesenteries play a key role in gut regeneration. The laboratory of Jose García-Arrarás has published some reports describing how during regeneration an intestinal primordium develops from a thickening of the mesenterial edge. With time this thickening grows up to the formation of a tube that will become the regenerated gut. One of the components of the sea cucumber gut is the enteric nervous system (ENS) that innervates the gastrointestinal tract. Now, a recent paper from this same laboratory describes for the first time in detail the regeneration of the enteric ENS in the sea cucumber Holothuria glaberrima (http://onlinelibrary.wiley.com/doi/10.1002/reg2.15/abstract).

Despite some studies have reported that miss-function of the ENS can cause several neuropathies both neurodegenerative and inflammatory in mammals, very few studies have analyzed the regenerative potential of this system under those contexts. Here, the authors use the sea cucumber to study how the ENS regenerates de novo together with the new gut after evisceration. In a first set of experiments, the authors use a collection of antibodies to label specific neural projections and cells of this ENS. Although, as the authors point out, it is not clear what are the antigens recognized by these antibodies, they are useful to consistently label specific neural fibers and cell populations and see how this pattern is restored during regeneration.

The main components of the ENS were divided depending on their location within the mesothelial layer, the connective tissue and the luminal layer. In the mesothelium they found a fiber network within the muscle layer that innervates the visceral muscle. There is a second mesothelial plexus formed by large nerve bundles that run along the longitudinal axis of the gut, and mostly parallel to the longitudinal muscle fibers. Within the connective tissue there are thick fibers that would correspond to nerves connecting the mesothelial plexus with the connective and luminal layers. There is also a network of small neurons and fine fibers all throughout this connective layer. Finally, the luminal plexus is formed by neuroendocrine-like cells distributed among the luminal epithelial cells. Occasionally, some of these cells display fiber projections extending towards the mesothelium.

Here, the authors used their markers to follow the regeneration process of all these components of the ENS at 3, 5, 7, 10, 14, 21, 28 and 35 dpe (days post evisceration). Summarizing all their stainings ENS regeneration could be divided into several stages: 1) initially there was a neurodegeneration stage consisting in the degradation of the preexisting fibers within the mesentery edge that will give rise to the intestinal primordium (5-7 dpe); 2) during the first days, then, the intestinal primordium lacked any ENS innervation (8-10 dpe); 3) then, re-innervation of this primordium started by 14 dpe. Here, new fibers appeared, mainly proximally in the mesothelium from an extrinsic source, that is, from cells in the mesentery. Moreover, new cells (of intrinsic origin) appeared also within the connective tissue, also mainly in proximal regions; 4) the fourth stage at around 21 dpe, was characterized by the differentiation of large fibers crossing from the mesothelium to the connective tissue as well as for the intrinsic differentiation of new neural cells within the lumen epithelium; 5) by 28 dpe most of the mesothelium was innervated and no differences between proximal and distal areas were observed; finally 6) by 35 dpe the ENS pattern in the regenerated intestine was similar to normal non-eviscerated intestine.

It is important to point out that ENS regeneration occurred in parallel to other important events. Thus, for example, the initial neurodegeneration coincided with the remodeling of the extracellular matrix. Also, fiber regeneration coincided with myogenesis and the incoming neural fibers were possibly re-innervating the newly differentiated muscle fibers. An important question to answer in future experiments concerns the origin of the new ENS cells. The authors discuss that they could originate from the dedifferentiation of muscle cells or coelomic epithelial cells or, alternatively from enteric stem cells or glia present in the intestine or mesentery. In the case of the neuroendocrine cells they seemed to differentiate from the luminal epithelial cells.

In summary, the authors describe here the different stages that characterize the regeneration of the ENS in sea cucumbers. As some events are conserved in mammals following lesions or inflammatory responses as, for example, the observed degeneration-regeneration stages, H. glaberrima can be a good model to understand ENS plasticity in other models. Moreover, there is an obvious interest for bioengineers trying to obtain intestines for transplantation and, as the authors state, studying ENS regeneration could provide insights into the type of cells and timing at which ENS precursors should be added in order to make a properly functional intestine.

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.

Francesc Cebrià

Francesc Cebrià

I am a Biologist and Professor at the University of Barcelona. I do my research on a fascinating animal: freshwater planarians. You can cut them in as many pieces as you want and each piece will regenerate a complete new flatworm in very few days. In this blog I will keep you updated on the latest news on the field of animal regeneration. You will be able to follow the latest research on how planarians, axolotls, newts, cnidarians and other animals are able to regenerate parts of their bodies

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