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.

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.

EMBO_regeneration_2014

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.

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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