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.

EMBO Regeneration Conference 2014

Back from the 2014 EMBO Conference on The Molecular & Cellular Basis of Regeneration & Tissue Repair held at St. Feliu de Guixols. This was an excellent meeting mainly focussed on basic aspects of regeneration in several animal models. The role of ROS signalling and the inflammatory response on wound healing and the triggering of a regenerative response were some of the hottest topics of the meeting, as well as neural and heart regeneration. Also, there were some talks and posters on new (or not so popular yet) models for regeneration such as the cnidarian Nematostella vectensis, several echinoderms and the crustacean Parhyale hawaiensis. It is also clear that large transcriptomic analyses and the enormous amount of data currently generated from RNAseq experiments in all different models and regenerative contexts will help to identify the molecular and cellular bases of key events of regeneration such us wound healing, blastema formation and growth, patterning and polarity, among others. Thus, comparative analyses might uncover novel conserved and taxon-specific elements required for regeneration. I hope we can discuss all this data in the near future in this blog.


Macrophages in zebrafish tail regeneration

In previous posts I have discussed the important role of macrophages and the immune system in triggering a successful regeneration in contrast to a scarring wound healing, by modulating the inflammatory response. Thus, for example, I commented on the requirement of macrophages to induce blastema formation during limb regeneration in salamanders probably by activating cell dedifferentiation and the proliferation of progenitor cells needed to rebuild the missing tissues (http://regenerationinnature.wordpress.com/2013/06/06/macrophages-and-limb-regeneration-in-salamanders/). Now, a paper from the laboratory of Timothy Petrie and Randall T. Moon reports on the need of macrophages also during tail regeneration in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24961798).

            Zebrafish tail regeneration may be divided into three main stages: 1) wound healing (0-1 days of regeneration); 2) blastema formation (1-3 days of regeneration); and 3) regenerative outgrowth and patterning of the new tissue (>3 days of regeneration). Previous studies in zebrafish larvae had reported that upon injury neutrophils and macrophages accumulate at the wound region suggesting a similar role of these cells compared to their mammalian counterparts. However, the role of these inflammatory cells in adult zebrafish regeneration remains mainly unknown.

            In this study, the authors used several transgenic lines to track neutrophils and macrophages upon amputation. For neutrophils, these cells rapidly accumulated at the wound from 6 hours post amputation (hpa). A maximum peak was achieved by 3 days post amputation (dpa) and then their number declined from 5 dpa until reaching pre-amputation levels of neutrophils by 7 dpa. It is known that regeneration rates are different along the proximo-distal axis of the tail. Thus, proximal amputations result in faster regeneration compared to slower growth upon distal amputations. Interestingly, proximal amputations recruited over twice the number of neutrophils as distal ones. Whereas few neutrophils were detected in uninjured tails, macrophages were found in higher density. Upon amputation macrophages began accumulating in the wound region by 3-4 dpa, reaching a peak by 6-8 dpa. As neutrophils, macrophages also accumulated faster and at greater densities in proximal amputations.

            Upon injury, neutrophils accumulation appeared to depend on their migration from the vasculature near the wound. The authors inhibited neutrophil recruitment into the wound but this reduced accumulation did not result in any difference in the rate of regeneration compared to controls. This is in contrast to what happens in larval tails as previous reports have suggested that neutrophil deficiency increases the regeneration rates. Next, the authors sought to determine the consequences of macrophage ablation during regeneration. They used a transgenic line bearing the enzyme nitroreductase (NTR) downstream of a macrophage-specific promoter. NTR converts the pro-drug metronidazole (MTZ) into a cytotoxic agent that kills the cell. Upon 36 h of MTZ treatment there was a strong reduction of around 80-90% of the macrophages in the tail. In control animals, MTZ by itself did not affect either the inflammatory response or the regeneration rate and success. Then, the authors amputated the tail and treated the animals with MTZ for 14 dpa. They saw that macrophage ablation resulted in a significant decrease of the extent of new tissue growth. Moreover, these fish also showed aberrant tissue growth along the regenerated tail. The zebrafish tail is composed, among other tissues, of segmented bony rays. Macrophage ablation resulted in a reduction in the average number of segments in the regenerated ray. Also, the degree of mineralization was reduced in NTR+MTZ fish, indicating that macrophage depletion impaired bone ray patterning and the quality of bone formation.

            Then, the authors analyzed in more detail which events required for regeneration could be affected upon macrophage ablation. They observed that although the loss of macrophages did not significantly affect gross blastema morphology and size, there was a significant decrease in cell proliferation. Also, at 4 dpa there was a strong reduction in the expression of regeneration-associated and injury-response genes. On the other hand, neutrophils normally accumulated upon macrophage ablation. In order to determine at what stage of regeneration are macrophages required the authors ablated them at two distinct time points. In one set of experiments they ablated them from 2 days before amputation through 3 dpa, corresponding to the stages of wound healing and blastema formation. The authors observed the same defects on regenerative rate and aberrant morphologies of the regenerated tails as when MTZ treatment was applied for 14 dpa. On the other side, and in order to analyze the requirement of macrophages during the outgrowth stage, the authors ablated the macrophages from 3 dpa through 14 dpa. In those experiments, the regeneration rate was not significantly affected; however, they still observed a higher occurrence of aberrant morphologies of the regenerated tails. So, it seems that during the early stages of regeneration macrophages would be required for blastema formation, whereas during tissue outgrowth macrophages would be also required to modulate tissue patterning.

            Finally, the authors wanted to study the relationship between the Wnt/b-catenin pathway and inflammation during tail regeneration as it has been shown that this pathway is required for blastema formation and outgrowth in zebrafish tail regeneration. Moreover, Wnt/b-catenin modulates several inflammatory processes in other models. Again, they used several transgenic lines to track the activation of this signaling pathway. As described above for neutrophils and macrophages, a greater density of cells with activated Wnt/b-catenin signaling were found in proximal amputations compared to distal ones. Flow cytometry analyses showed that less than 1% of neutrophils and 3% of macrophages exhibited activated Wnt/b-catenin signaling. Interestingly, macrophage accumulation at the wound was almost completely inhibited after inhibiting Wnt/b-catenin. Also, the inhibition of this pathway resulted in delayed neutrophil resolution and prolonged neutrophil number in the wound region, suggesting that the Wnt/b-catenin pathway might be required for the progression of the injury response after amputation. Thus, Wnt signaling might mitigate the initial inflammatory response and function as a molecular switch from neutrophil resolution to macrophage enrichment.

            In summary, this study reports on the requirement of macrophages for zebrafish tail regeneration providing a functional link between inflammation and regeneration.

Summer break

Just a short notice to inform you that I am going to take some weeks off for summer break. I will back in September to continue posting about regeneration. The beginning of September will be also an exciting time as there will be the EMBO Conference on Regeneration in Sant Feliu de Guixols (near Barcelona), so I hope we can learn a lot from the data presented there.

Have a nice summer!

Neoblast heterogeneity and planarian regeneration

Compared to other regeneration models, freshwater planarians display a distinctive feature: they regenerate from a unique population of adult pluripotent stem cells, called neoblasts. This makes planarian an excellent model in which to study the behaviour of stem cells in vivo. Although classically seen as a rather homogeneous cell population, several recent studies have suggested that neoblasts could, in fact, constitute a wide heterogeneous population, based on different parameters and features, in which, for instance, several progenitor-like cells for different cell types exist. Now, a beautiful paper from the laboratory of Peter Reddien goes much deeper into their analysis of planarian stem cells and identifies two major and functionally distinct cellular compartments among neoblasts (http://www.ncbi.nlm.nih.gov/pubmed/25017721).

In this study the authors characterized the neoblasts at a single-cell resolution level. Neoblasts are the only proliferative cells in planarians. Fluorescence-activated cell sorting (FACS) can be used to isolate proliferating neoblasts based on their DNA content. The authors then analysed the expression of 96 genes within each of hundreds of individual neoblasts. These genes were selected from a neoblast transcriptome and included, among others, well-known neoblasts and post-mitotic markers, as well as a variety of transcription factors and regulators highly abundant in neoblasts. Hierarchical clustering allowed distinguishing two main classes of equally sized populations of neoblasts: the zeta-class and the sigma-class. The zeta-class neoblasts were characterized by a high expression of zfp-1, g6pd, fgfr-1, p53, soxP-3 and egr-1. On the other hand, the sigma-class neoblast showed low expression levels of the previous genes and high expression of a distinct set of genes: soxP-1, soxP-2, soxB-1, smad6/7, inx-13, pbx-1, fgfr-4 and nlk-1.

In order to discard that these two neoblast populations could be in fact a single population but at different state of the cell cycle the authors isolated by FACS different neoblast populations at different stages of the cell cycle according to their DNA content. Single-cell profiling showed that both classes, zeta- and sigma-neoblasts, were equally present throughout the cell cycle. Also, zeta- and sigma-neoblasts showed a similar broad spatial distribution along the planarian body. Next, the authors checked how these two populations responded to either amputation (anterior or posterior regeneration) or sublethal irradiation. In all cases, after two days of amputation or irradiation the relative abundances of the zeta- and sigma-classes were the same as in control, untreated animals.

Upon amputation, neoblasts display a bimodal proliferating response with a first mitotic peak at 6 hours (all throughout the regenerating fragment) and a second peak at 48 hours (localized at the wound region). Remarkably, the sigma-neoblasts were overrepresented among the mitotic population both at 6 and 48 hours, indicating that these two mitotic peaks derived mainly from the activity of the sigma-neoblasts. Moreover, analyses on the spatial distribution of these neoblasts indicated that the accumulation of neoblasts at the wound region at 48 hours of regeneration depended mainly on the sigma-class neoblasts.

Next, the authors focussed on the functional analysis of zfp-1, a gene specific to the zeta-class. Upon its silencing by RNAi, the animals died in few weeks. Zfp-1 RNAi resulted in the loss of expression of other zeta-class markers without affecting the expression of sigma-class genes. In fact, the silencing of zfp-1 eliminated the zeta-class neoblasts without affecting the sigma-class cellular compartment. In terms of function, the depletion of the zeta-class neoblasts did not interfere with the normal behaviour of the sigma-class as these cells could mount a proper regenerative response generating two mitotic peaks at 6 and 48 hours as in controls. As the animals in which zfp-1 was silenced were still capable of regenerating a blastema the authors checked whether specific cell lineages were affected in those animals. Remarkably, they found that brain, gut, muscle, protonephridia, eyes and pharynx tissues were apparently normal, indicating that the sigma-class neoblasts were capable of differentiating into a broad range of cell types. However, other cells characterized by the expression of previously identified as neoblast early and late progeny markers were depleted. These genes included prog-1 (early progeny) and AGAT-1 (late progeny). These genes label subepidermal cells that undergo rapid cell turnover. However, it was not clear whether those cells defined a particular cell lineage. Here, by doing RNAseq, the authors found out that several transcripts associated with epidermis, cilia and secretory cells were reduced after the silencing of zfp-1. Specifically, they identified nine genes expressed subepidermally (as prog-1) and seven genes expressed at the epidermis, and whose expression was clearly affected after zfp-1 RNAi. Consequently, the epidermal cells were disorganized, thinner and less abundant in those animals. Moreover, after two weeks of BrdU incorporation much fewer epidermal cells were labelled with BrdU, suggesting that zeta-class neoblasts gives rise, at least in part, to an epidermal cell lineage, probably through an intermediate stage defined by the expression of prog-1 and AGAT-1.

Finally, and through some elegant experiments of transplantation of sigma-class neoblasts into previously irradiated (and therefore depleted of any neoblast) planarians, the authors showed how the sigma-class neoblast were capable of generating the zeta-class compartment. Then, and by checking the gene expression profile of individual cells at different stages of the cell cycle, the authors suggest that zeta-class neoblast would derive from sigma-class cells just following their entry into S phase.

In summary, this study identifies two clearly distinct classes of neoblasts based on their gene expression profiles, response to wounding and cell differentiation potential. The data presented here also indicates that these two classes can be also rather heterogeneous themselves. Thus, for instance, within the sigma-class the authors suggest the existence of a subclass, the gamma-neoblast, characterized by the expression of some genes that had been previously related to the planarian gut, indicating that those gamma-class cells could be related to the gut lineage.

JNK balances cell death and proliferation during planarian regeneration and remodeling

In any developmental system cell proliferation and cell death need to be tightly regulated to ensure proper growth, morphogenesis and patterning. During regeneration these cellular processes must be also coordinated in order to achieve a well-proportioned animal de novo upon regeneration completion. A recent paper from the laboratory of Emili Saló and Teresa Adell (http://www.ncbi.nlm.nih.gov/pubmed/24922054) reports on the key function of the JNK pathway in regulating these events in regenerating and degrowing planarians. JNK is a stress-activated protein kinase belonging to the MAPK family that, in other systems, has been implicated in the regulation of cell cycle, wound healing, neurodegenerative disorders and cancer.

Planarian JNK is expressed in the central nervous system as well as in the neoblasts (planarian adult pluripotent stem cells). Upon silencing of this gene by RNAi, regeneration was severely inhibited as the treated animals regenerated very small blastemas with aberrant differentiation of the new structures within them. JNK RNAi did not affect the early expression of the polarity determinants notum and wnt1, at those stages in which polarity is re-established. However, the latter expression of these genes was significantly attenuated indicating that JNK is somehow required for the maintenance of the expression of such polarity genes. Whereas in other systems such as Drosophila the JNK pathway is required for wound closure, this was not affected after JNK silencing in planarians. However, JNK RNAi resulted in the failure to activate the expression of several wound-induced genes.

Upon amputation neoblast proliferation dynamics displays a bimodal response. There is a first proliferative peak at 6 h after amputation that is systemic throughout all the regenerating pieces, and that it has been associated to wounding. A second mitotic peak is seen at 48 h of regeneration concentrated around the wound region; this second peak is associated to tissue loss and the regenerative response that leads to blastema formation. JNK RNAi does not affect the number of neoblasts or the proportion of actively cycling cells. However, the authors observed that the first mitotic peak was elevated and the second peak occurred about 10 h earlier than in controls. CldU labeling combined with piwi1 (a neoblast-specific marker) and phosphohistone H3 (a marker of the entering to the M phase of the cell cycle) suggests that JNK RNAi induces the shortening of the G2 phase of the cell cycle and, therefore, neoblast enter faster into mitosis. This shortening of the G2 did not affect the capacity of those neoblasts to give rise to normal numbers of post-mitotic progeny.

In addition to neoblast proliferation and blastema formation, the pre-existing tissues must go through a remodeling process so the regenerated animal achieves proper body proportions. This remodeling is largely dependent on cell death. After amputation apoptotic cell death, in planarians, follows also a bimodal response with a first apoptotic peak at 4 h post amputation concentrated at the wound region, and a second apoptotic peak at 3 days of regeneration systemically found throughout the entire regenerating fragment. This second apoptotic peak has been associated to the remodeling of the pre-existing tissues. JNK RNAi inhibited these two apoptotic peaks. Remarkably, the inhibition of apoptosis was accompanied by an increased proliferation in those pre-existing tissues that need to go through remodeling. As a consequence, these treated animals were incapable of readjusting the position of pre-existing organs such as the pharynx to restore proper body proportions. Overall, these results indicate that JNK is necessary to trigger a proper apoptotic response.

During planarian regeneration the second mitotic and apoptotic peaks are related to tissue loss whereas the first peaks are related to a general systemic response to wounding. Thus, after a wound that does not imply tissue loss only the first mitotic and apoptotic peaks are observed. Interestingly, JNK RNAi did not affect neither the wound-associated proliferative and apoptotic responses or the normal expression of wound-induced genes. In contrast, the proliferative and apoptotic response after small injuries that imply loss of small amounts of tissues depended on the function of JNK, as it happens upon amputation of large regions. Therefore, JNK is required for regeneration in those contexts in which tissue has been lost.

In addition to their amazing regenerative capabilities, planarians are very plastic animals as they constantly grow and degrow depending on food availability. Planarian growth and degrowth depend upon the balance of cell proliferation and cell death. Again these two cellular processes must be tightly regulated as animals keep proper body proportions at any time. In starved animals that consequently will degrow, the silencing of JNK inhibits apoptosis without affecting the proliferation rates. This inhibition of apoptosis is accompanied by the impairment of proper body re-scaling during degrowth. Remarkably, JNK RNAi did not affect the apoptotic response in growing animals and they underwent through proper body re-scaling during their growth. Therefore, the authors conclude that JNK is required for the apoptotic-driven remodeling that takes place in degrowing animals to maintain proper body proportions.

In summary, JNK is required to trigger a proper regenerative response after any wound that results in tissue loss. There, JNK is required to induce apoptosis, regulate the onset of mitosis in neoblasts and trigger the expression of wound-induced genes. Moreover, in intact starved animals that are degrowing JNK is necessary to induce the apoptotic driven tissue remodeling and rescaling of proper body proportions.

Wnt and BMP pathways coordinate bone regeneration in zebrafish

Several weeks ago I commented on a study from the laboratory of Gilbert Weidinberg in which they had characterized an organizing center defined by the Wnt/b-catenin pathway within the distal blastema of regenerating zebrafish fin, that would control regeneration by regulating the function of several downstream signaling pathways that would mediate the effects of this organizer on surrounding tissues. Here, I comment on a study from the laboratory of Scott Stewart and Kryn Stankunas that describe how the Wnt/b-catenin and BMP signaling pathways work together and in opposite directions to coordinate bone regeneration during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24485659).

In zebrafish, bone regenerates through dedifferentiation and re-differentiation of lineage-restricted osteoblasts. Osteoblasts are the responsible of depositing the osteoid, a unique extracellular matrix that form the mature bone. Although previous reports have implicated several signaling pathways, including Wnt/b-catenin and BMP receptor, in this process, how they act at the cellular and molecular levels to drive a successful regeneration is not completely known. Here, the authors first analyzed the expression of Runx2 and sp7, two transcription factors with well-known roles on bone formation. Early after amputation Runx2 was upregulated in osteoblasts lining preexisting bone adjacent to the amputation plane. Later, some Runx2+ mesenchymal cells expressed also sp7. Then, Runx2-/sp7+ cells first appeared near the amputation plane. By 72h of regeneration the osteoblast lineage was highly organized along the proximo-distal axis of the blastema: Runx2+ cells were located in most distal regions while sp7+ cells were mainly found near the amputation plane. In between Runx2+/sp7+ cells were found. In terms of proliferation, more Runx2+ cells incorporated EdU compared to sp7+ cells, suggesting that sp7+ cells near the amputation plane would be non-proliferative osteoblasts that append to progressively elongating bone.

After amputation, osteoblasts dedifferentiate to give rise to Runx2+ preosteoblasts. The authors showed that osteoblasts have epithelial-like properties as they were labeled with antibodies against catenins (a- and b-) that are found in adherens junctions that interconnect epithelial sheets. During regeneration, distal Runx2+ did not express a-catenin in contrast to Runx2+/sp7+ and sp7+ cells in close proximity to new bone, that were positive for membrane-localized a-catenin. At 24h after amputation osteoblasts rapidly lost a-catenin expression as they dedifferentiate into a progenitor state. Moreover, as they became Runx2+ and Runx2+/sp7+ cells they changed their shape from long and thin to a more compact, polygonal morphology. These results suggested that osteoblast went through an epithelial-to-mesenchymal transformation (EMT) during regeneration. This was further supported by the observation that twist2, a well-known transcription factor that directs EMT, and runx2a were rapidly induced in tissue adjacent to the amputation plane. Later, distal Runx2+ cells co-expressed twist2. Therefore, the authors concluded that Runx2+ cells originated from EMT of differentiated osteoblasts and distal Runx2+ preosteoblasts were maintained in a mesenchymal twist2-expressing state.

Next, the authors analyzed the Wnt/b-catenin signaling in regenerating fins. At 24h of regeneration, Runx2+ cells had nuclear b-catenin staining. By 72h post amputation, strong nuclear b-catenin was observed in distal Runx2+ preosteoblasts with much less staining in sp7+ differentiating osteoblasts near the amputation plane, suggesting that downregulation of Wnt signaling correlates to osteoblast maturation. It is known that Wnt signaling can initiate EMT and induce twist expression during mouse bone development. Here, the authors used IWP-2, an inhibitor of Wnt signaling. IWP-2 treatment arrested regeneration by interfering with osteoblast EMT and the induction of twist2 expression, indicating an important role of Wnt signaling in osteoblast EMT. Moreover, IPW-2 treatment from 48h to 72h post-amputation also blocked regeneration by depleting osteoblast-lineage cells distal to the amputation plane, suggesting a role of this pathway in maintaining the preosteoblast population.

Finally, the authors analyzed the BMP pathway as it has been also implicated in bone formation. The activation of the BMP signaling leads to the phosphorylation of the transcription factor Smad1/5/8, that can then go to the nucleus and activate its downstream target genes. Here, pSmad1/5/8 was detected in differentiating sp7+ cells but not in Runx2+ or Runx2+/sp7+ preosteoblasts. The inhibition of BMPR resulted in a pronounce decrease in the extend and levels of sp7 expression and reduced bone formation. These treated fins were able to form a blastema but failed to produce mineralized bone, suggesting a role of the BMP pathway in osteoblast maturation. Remarkably, BMPR inhibition resulted also in an increase in the number of Runx2+ cells and a decrease of the Runx2+/sp7+ and sp7+ populations. Osteoblast proliferation and cell death were not affected by this treatment suggesting that BMP would drive osteoblast differentiation.

As BMP inhibition expanded proximally the distal Runx2+ population the authors hypothesized that BMP activity in proximal regions would normally inhibit Wnt/b-catenin activity in those proximal domains. This was supported by the observation that BMPR inhibition reduced the expression of Dkk proteins, well-known negative regulators of Wnt activity. In agreement with the idea of distal Wnt active and proximal BMP active populations, wnt5a and wnt5b were mainly expressed at the distal tip of the blastema whereas bmp2 was expressed in differentiating proximal osteoblasts.

In summary, the authors have shown that zebrafish bone regeneration is mainly regulated by the antagonistic and coordinated function of the Wnt and BMP signaling pathways in order to provide a precise balance between cell plasticity and differentiation. In their proposed model, Wnt activity drives EMT of osteoblasts to give rise to dedifferentiated Runx2+ preosteoblasts. Sustained levels of Wnt activity in the distal blastema maintain these Runx2+ proliferative cells. Then, as these preosteoblasts are located to more proximal regions they upregulate bmp2 and activate autocrine BMP activity that promotes osteoblast differentiation by inducing the expression sp7 and dkk1b, that inhibits Wnt activity to prevent the overexpansion of the progenitor pool.

V-ATPase activity during adult zebrafish fin regeneration

In recent years several papers have uncovered the importance of ion channels during regeneration in different models. Thus, for example, cellular hyperpolarization is essential for Xenopus tadpole tail regeneration and cellular depolarization is required to specify anterior polarity in planarians. Now a recent paper from the laboratories of Ana Certal and Joaquín Rodríguez-León has reported for the first time the requirement of V-ATPase activity for fin regeneration in adult zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/24671205).

After any wounding an electric current is generated as a response; however, only in those cases in which a regenerative process is triggered these endogenous electric currents are maintained beyond wound closure. Here, the authors first analysed the contribution of different ions (K+, Na+, H+, Ca2+ and Cl-) to the electric current during adult zebrafish fin regeneration. Of all these ions H+ was the only one for which the authors found that 24 hpa (hours post-amputation), during blastema formation, there was still an efflux that was 14-fold higher than the one detected in intact fins. Previous microarray experiments had detected V-ATPase, a main H+ transporter, as being upregulated after 24h during fin regeneration. Thus, the authors checked the expression of several V-ATPase subunits during regeneration. Two of them, atp6v1a and atp6ve1b, were not expressed in intact fins but were upregulated in the blastema by 24 hpa. At 72 hpa some expression was still detected at the distal part of the blastema. Next, the authors assessed the role of V-ATPase during regeneration. They blocked the pump’s activity either by using concA or morpholinos (MO) against atp6v1e1b. Both approaches delayed fin regeneration suggesting a role for this H+ pump in the regenerative process.

As it happens for amphibian limb regeneration, proximal amputations of the caudal fin resulted in higher regeneration rates compared to distal stumps. Interestingly, the expression of atp6v1e1b was already visible by 12 hpa, whereas in distal stumps the first expression of this gene was observed at 24 hpa. Not only the expression appeared earlier but it also covered a wider region. By 48 hpa these differences were not so obvious anymore. In agreement with this earlier and stronger upregulation of atp6v1e1b in proximal stumps, the authors found out that the H+ efflux started earlier in those proximal stumps (3 hpa instead of 12 hpa in distal ones) and was higher at any time point measured than in distal stumps. These results clearly indicate a relationship between V-ATPase and H+ efflux and the regeneration rate along the PD (proximo-distal) axis. Further supporting this, atp6v1e1b knockdown significantly decreased the H+ efflux. This MO-mediated silencing of atp6v1e1b also resulted in a decreased regenerated area, being this inhibition higher in proximal stumps, suggesting that those proximal stumps with higher regenerative rates are more dependent on V-ATPase activity. Remarkably, the inhibition of V-ATPase activity did not seem to affect the regeneration of the larval fin fold.

Finally, the authors studied the effects of inhibiting the V-ATPase activity on cell proliferation and gene expression. Although no differences in proliferation were observed at 24 hpa, by 48 hpa atp6v1e1b knockdowns showed a significant reduced number of proliferative cells within the blastema, compared to controls. Different signalling pathways, including FGF, Wnt/B-catenin and Retinoic acid (RA), have been shown to regulate cell proliferation during regeneration. In controls, the expression of mkp3 (FGF signalling) and aldh1a2 (RA signalling) was detected in wider domains in proximal stumps compared to distal ones, similarly to the differences observed for V-ATPase activity. The silencing of atp6v1e1b resulted in the inhibition of the expression of mkp3 and aldh1a2 indicating that V-ATPase was required for the expression of these two genes during regeneration. Last, the authors reported that V-ATPase seems to be also necessary for the normal innervation of the regenerating fin.

In summary, this study reports for the first time the requirement of V-ATPase for adult zebrafish fin regeneration. The authors propose that the regulated H+ efflux generates pH and/or voltage domains within the regenerating tissue that, directly or indirectly (for example, via innervation), would act on FGF and RA signalling pathways to regulate cell proliferation during regeneration.

Wound healing in injured and regenerating Nematostella

Wound healing is a universal response to injury conserved in all animals. However, not in all cases wound healing is followed by a successful functional regeneration. Thus, for example, skin injuries in adult mammals are usually solved through a so-called scarring wound healing that does not allow a functional recovery of the damaged skin. On the other side, in regeneration-competent species wound healing does not a have a negative effect but on the contrary is a key first step to trigger a regenerative response. Thus, in those cases, impairing wound healing results in the inhibition of regeneration. A deep characterization of the cellular and molecular events that result in scarring or regenerative wound healing may be very important to try to develop strategies and therapies to enhance the poor regenerative abilities shown by many animals. Now, a recent paper from the laboratory of Mark Martindale has characterized the regenerative wound healing in the cnidarian Nematostella vectensis (http://www.ncbi.nlm.nih.gov/pubmed/24670243).

In a first set of experiments the authors characterized the cellular and molecular events that occurred after injuring the animals by making punctures in their bodies with a glass needle. Two hours after injury an enrichment of actin was seen around the injury site and the wounds were healed after 6 hours. In another cnidarian, Hydra, and some vertebrates, apoptosis is required to trigger a proliferative response that leads to a successful regeneration. Similarly, upon injury along the ectodermal surface of Nematostella, apoptosis was significantly upregulated. Next, the authors decided to conduct a pharmacological screen to see which signaling pathways could have a role in wound healing and regeneration in these animals. Inhibition of the Notch pathway blocked head regeneration without affecting wound healing. On the other side, and unexpectedly, they did not found any defect after blocking the TGFB signaling. Finally, they inhibited ERK signaling and found a strong impairment of wound healing and regeneration. The MAPK signaling pathway plays many functions including immune response, cell proliferation, apoptosis and cell movement. In Drosophila, ERK (through MAPK) regulates actin dynamics at the injury site a the early stages of wound healing. Using their puncture assay they found that inhibiting ERK signaling with the drug U0126 caused wound to remain open after six hours and also eliminated the local phosphorylation of ERK at one hour after injury, compared to the wound response of untreated animals. U0216 did not blocked the initial apoptotic response to injury indicating that apoptosis by itself is not sufficient to trigger a regenerative response. Also, the animals treated with U0216 did not show much actin concentration around the injury suggesting that ERK could be targeting cell movement and adhesion.

Then, the authors used Nematostella genome-wide microarrays to identify genes involved in wound healing. They analyzed the gene profiles from samples taken 1 hour and 4 hours after injury in untreated and U0216 treated animals, which allowed them to do many comparisons. Thus, they generated a profile of genes not only up- or down-regulated at early (1h) and late (4h) stages of normal wound healing, but also how the expression of those genes was affected after inhibiting the ERK pathway. After injury and wound healing genes upregulated included genes with peptidase activity, modulators of MAPK signaling, Sox E1 and runt transcription factors, growth factor-related genes as well as genes related to mucus proteins. Some of these genes were validated by qPCR and/or in situ hybridizations. The authors focused then in several genes: uromodulin, soxE, thiamine enzyme, a matrix metalloproteinase (MMP) inhibitor and a maltase-like gene. In all cases these genes were upregulated upon injury and this upregulation appeared dependent of ERK signaling, as it was not observed after treatment with U0216. Remarkably, all these genes were upregulated during regeneration after amputation through the oral-aboral axis. Again, the expression of these genes during regeneration was dependent of ERK signaling.

To conclude, the authors propose that ERK signaling would be necessary for the initiation of the early wound healing response in Nematostella, agreeing with the important functions of the ERK signaling during regeneration reported in other systems. Future functional analyses on the genes identified here should help to confirm this hypothesis and to better characterize wound healing at the gene expression level. In summary, this is the first report of genes involved in wound healing in Nematostella. Comparisons of the cellular and molecular events that characterize Nematostella wound healing with those found in other regenerative models as well as in regeneration-incompetent animals could help to understand better this key initial process that takes place after any injury.

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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