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A hematopoietic cell-derived signal is required for cell survival during zebrafish fin regeneration

During zebrafish fin regeneration different genes and signalling pathways are locally upregulated in the wound area and play important roles to trigger a successful regenerative response. However, less is known about the existence and function that putative factors provided by tissues away from the wound could have during these regeneration events. A recent paper from the laboratory of Atsushi Kawakami reports on a diffusible signal derived from hematopoietic cells that would support cell survival and proliferation during zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25533245).

The authors studied a mutant called cloche (clo) previously identified from a phenotype that lacks most hematopoietic and endothelial cells. In wild-types (fish larvae), the fin fold was normally regenerated (in size and shape) by 3 days after amputation. In contrast, clo mutants displayed aberrant regeneration with the formation of tissues with a blister-like appearance. The authors thought that this might represent dying cells so they checked with TUNEL assay to find out a large number of dying cells in this region, compared to wild-types. Uncut clo mutants did not display higher levels of cell death so it seemed that this increase in cell death in clo mutants was regeneration-dependent. This was further confirmed because in a wound healing assay there was no increase in cell death in these mutants. This increase in cell death was accompanied by a decrease in cell proliferation as assayed by BrdU labelling. Again, here, the cell proliferation levels in the head and trunk (away from the wound) were normal compared to wild-types.

Immediately after amputation (0,5 hours), necrotic cell death was similarly induced in the injured region of wild-types and clo mutants. This necrotic cell death disappeared by 6 hours in wild-types and clo mutants, but then at 12 hours after amputation clo mutants displayed a high number of TUNEL positive cells. This cell death was mostly suppressed when overexpressing Bcl2 (a negative regulator of apoptosis) in these mutants, suggesting that cell death was most probably caused by a Bcl2-regulated apoptosis pathway.

In clo mutants this increased apoptosis coincided with the stage in which regenerative cell proliferation begins. The authors checked then the expression of junb and junbl early markers induced in the wound epithelium and the blastema, respectively. These two genes appeared to be normally induced in the clo mutants, suggesting that the initial regenerative response might be normal in them. Next, they carried out some transplantation experiments between clo mutants and wild-types to investigate the cell autonomy of Clo protein function during regeneration, concluding that Clo function was non-cell autonomous for the survival of the regenerative cells. These results prompted them to wonder whether the lack of hematopoietic cells in clo mutants was responsible for these reduced cell survival during regeneration. Thus, they analysed tal1, another hematopoietic mutant, and found out similar phenotypes: impaired fin fold regeneration with increased apoptosis and reduced cell proliferation. As the hematopoietic lineage contains several cell types they wanted to identify the cell types required for the survival of the regenerative cells in these mutants. In red blood and endothelial cell mutants they did not observed apoptosis of the regenerative cells. However, when they knocked down the myeloid lineage through the silencing of pu.1, a transcription factor required for myeloid cell differentiation, they obtained similar results to those observed in clo and tal1 mutants, suggesting that myeloid cells would have an important function on the survival of the regenerative cells.

Finally, they wanted to investigate how this putative signal was mediated from the hematopoietic tissues to the amputation site. They developed an in vitro assay for a tail explant cultured for 12 hours post-amputation. Wild-type and clo and tal1 mutant explants normally induced the expression of junb and junbl. Whereas in wild-types no apoptosis was observed, explants from clo and tal1 mutants displayed an induced cell death. These results suggest that the defects observed in clo mutants could not be caused by an impaired circulation. Remarkably, when the mutant explants were treated with body extracts prepared from wild-types there was a rescue of the apoptosis. On the other hand, body extracts from the mutants did not rescue the apoptosis in the tail explants from those mutants. These results suggested that the survival of the regenerative cells might be supported by a diffusible factor in the wild-type body.

In summary, this study suggests that a diffusible, and still unknown, factor derived from the hematopoietic cells would support the survival and proliferation of primed regenerative cells during fin fold regeneration in zebrafish.

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.

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.

The role of the Hippo pathway in Xenopus limb bud regeneration

As I have discussed before in this blog, the Hippo signalling pathway has a conserved function in controlling organ size and patterning through the regulation of the balance between cell proliferation and cell death. During regeneration these processes must be tightly regulated so the regenerated organs and structures attain proper sizes. However, not much is known yet about the role of Hippo signalling during regeneration. Previous reports have shown that this pathway is required for proper insect leg as well as Macrostomum (flatworm) regeneration. In fact, for Macrostomum, I discussed those results in this blog (https://regenerationinnature.wordpress.com/2013/06/27/the-hippo-pathway-in-macrostomum-homeostasis-and-regeneration/).

Now a recent paper from the laboratory of Hitoshi Yokoyama reports a role for Yap1 during regeneration of the Xenopus limb bud (http://www.ncbi.nlm.nih.gov/pubmed/24491818). The transcriptional co-factor Yap1 (Yorkie in invertebrates) is the canonical effector of the Hippo signalling pathway. When the Hippo pathway is activated, Yap1 gets phosphorylated and becomes inactive as it gets trapped in the cytoplasm. On the other side, active dephosphorylated Yap1 can enter the nucleus where it activates the expression of its target genes. In other systems it has been shown how the over activation of Yap1 (or Yorkie) leads to an increase of proliferation and a decrease of apoptosis. Conversely, the inactivation of Yap1 results in decreased cell proliferation and higher levels of apoptosis.

In this study, the authors analysed the regeneration of amputated limb buds of Xenopus tadpoles at stage 52. In a first set of experiments they found that all the main core components of the Hippo pathway, including some regulators and target genes, were expressed in 5-day blastemas. As Yap1 is the effector of the pathway they focussed on this gene. In situ hybridizations showed that Yap1 was upregulated within the blastema. Taking advantage of the cross-reaction of an antibody against human Yap1 they could see how in intact limb buds Yap1 was in the cytoplasm whereas, upon amputation, Yap1 was localized in the nuclei of blastema cells. These results suggested that Yap1 was translocated to the nuclei during regeneration where it could activate its target genes. Next, the authors characterized the function of Yap1 during regeneration by using a dominant-negative form of this gene (dnYap) under control of a heat shock promoter. Overexpression of dnYap resulted in impaired limb regeneration characterized by a reduction in the size and number of skeletal elements, as well as a reduction in the number of digits in those regenerated limbs. Interestingly, the overexpression of dnYap did not apparently affect the normal development of the uncut contralateral limb bud.

In addition to these external defects, the authors checked the expression of several patterning genes with well-defined and region-specific patterns. The overexpression of dnYap impaired the expression of hoxa13 and hoxa11 so both genes partially overlapped in contrast to their expression in well-separated domains in controls. Also, the expression of shh, fgf8 and mkp3 was clearly downregulated. Moreover, the small blastemas of dnYap animals contained much less differentiated muscle and innervation. As the Hippo pathway controls cell proliferation and apoptosis the authors checked how these events were affected after overexpressing dnYap. The number of mitotic cells significantly decreased within the blastema as well as in the stump. On the other side, ectopic apoptotic cells were found the stump. These results suggested that the impaired regeneration phenotype was associated to missregulation of cell proliferation and apoptosis. Again, the overexpression of dnYap did not affect cell proliferation, apoptosis or differentiation in uncut limb buds and they developed into normal limbs.

In summary, this paper shows how Yap1 is required for limb bud regeneration in Xenopus. In this context Yap1 appears to have a conserved role in the regulation of cell proliferation and apoptosis. Future experiments should determine whether other elements of this pathway as well as its upstream regulators are equally conserved between vertebrates and invertebrates and are also required for regeneration.

Autophagy in zebrafish fin regeneration

Autophagy is a biological process through which cells self-degrade unnecessary or dysfunctional components. Autophagy may be important in certain contexts such as for example nutrient starvation where a minimum level of energy is required to maintain the basic functions that warranty the survival of the cells. Another context in which autophagy can be important is during the remodelling of the cytoplasm that takes place in the process of cell reprogramming. In animal models in which regeneration depends upon an initial dedifferentiation of differentiated cells into proliferative cells, an extensive cytoplasmic remodelling must occur. However, few data is available about the role that autophagy may play during regeneration. Now, a recent paper from the laboratories of DJ Klionsky and T Vellai reports on the role of autophagy in zebrafish fin regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24317199).

The authors use first a transgenic line carrying a GFP reporter under the control of Lc3 promoter, a specific marker for autophagosomes and autolysosomes. They found that Lc3 was upregulated within the tail fin blastema at 2 days after amputation (dpa). From that time the levels of Lc3 gradually decreased until reaching basal levels around 6 dpa. Lc3 was expressed in newly differentiated cells within the blastema. As those cells come from dedifferentiation, Lc3 expression could be reflecting an autophagic activity during this dedifferentiation-redifferentiation process. This increase in autophagy during regeneration was further corroborated by observations of electron microscopy. Thus, an increase of autophagic features was detected in epidermal cells, osteocytes and pigment cells of 2-day blastemas.

In order to see whether this increase in autophagy had a role during regeneration the authors used different methods to block this process. They either injected an antisense morpholino oligonucleotide against atg5 or a drug that acts as an autophagy inhibitor. Silencing of atg5 at 2 dpa blocked regeneration and induced the degeneration of the existing blastema. Similar results were obtained after using the inhibitory drug. Because in other models the inhibition of autophagy induces apoptosis the authors checked next whether the defects in regeneration after blocking autophagy could be explained in terms of increased apoptosis. Indeed, an increase of apoptotic cells within the blastemas was observed. Not only apoptosis was missregulated but also they observed a significant reduction of proliferative cells as well as problems with cell differentiation. All these data lead the authors to conclude that autophagy would promote cell survival and proliferation and would mediate cell differentiation during regeneration.

Finally, the authors looked for upstream regulators of autophagy in this context. It is well known that the FGF signalling is required for fin regeneration and it silencing inhibits cell dedifferentiation. MAPK/ERK is one of the downstream effectors of FGF signalling and has been involved with the regulation of autophagy. Therefore, the authors checked whether the blocking of MAPK/ERK itself influenced fin regeneration. Using an inhibitory drug they found a complete impairment of fin regeneration. Remarkably, they also observed a decrease of autophagy in those treated animals, suggesting that MAPK/ERK activity was required for the upregulation of autophagy during regeneration. From all that data the authors propose a model in which Fgf signalling would lead to the phosphorylation of MAPK/ERK that would then regulate autophagy.

In summary, the authors conclude that autophagy would act as a prerequisite for the regeneration of the zebrafish tail fin through the regulation of the reorganization and remodelling of the cytoplasmic compartment during cell dedifferentiation.

ROS species, apoptosis and adult caudal fin regeneration in zebrafish

In previous posts I have discussed the positive role that ROS species have during tail regeneration in Xenopus as well as the important role played by apoptosis to trigger head regeneration in Hydra. Now a recent paper from the laboratory of Sophie Vriz links both processes during adult caudal final regeneration in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/23803955).

First, the authors show how whereas wounding induced a transitory ROS production with a peak 30 minutes post-lesion that disappeared after 2 hours, fin amputation induced a much prolonged production of ROS peaking at about 12-16 hours post-amputation (hpa). ROS production was not detected after 24 hours of regeneration. Then, and to try to see the functional relevance of this regeneration-specific extended ROS production the authors used different inhibitors of ROS. Thus, inhibiting NOX (NADPH oxidase) activity from 12 to 24 hpa seemed sufficient to reduce the size of the regenerated fin at 72 hours. This reduced regeneration was accompanied by a decrease in the expression of klf4 (a progenitor cell fate marker) and dio3 (involved in progenitor cell proliferation). The expression of another progenitor cell fate marker as it is myc was not affected.

As oxidative stress has been related to MAP kinase activation and apoptosis, the authors wanted to search whether ROS was also involved in those processes during caudal fin regeneration. First, they observed that the normal activation of the JNK pathway at 6 hpa was reduced about at 50% after NOX inhibition, suggesting that ROS has a role in the early activation of the JNK pathway. Second, the inhibition of ROS production reduced also the number of apoptotic cells during the first 18 hpa. Then, the authors checked in more detail the cell death and apoptotic responses after amputation. Remarkably they found a bimodal response with two peaks of TUNEL positive cells (also active caspase-3 positive cells) at about 5 hpa and 15-18 hpa mainly concentrated in the stump epithelium and very few cells in the mesenchyme. In contrast, after wounding, and in agreement with the only early peak observed for ROS production, cell death increased and peaked around 6 hpa without a second peak later. To further characterize the function of apoptosis on regeneration the authors inhibited this process during fin regeneration. Remarkably, blocking the 2nd wave of apoptosis (from 12 to 72 hpa) was sufficient to impair regeneration. In fact, inhibiting the first apoptotic wave (from 4 to 10 hpa) neither blocked regeneration nor affected the 2nd apoptotic wave. These results suggest that the 2nd apoptotic wave was the one required for regeneration. However, these effects on regeneration affect only the size of the regenerate because those small blastemas appeared to be normally patterned.

Next, the authors looked how proliferation was affected after JNK and apoptosis inhibition. In control animals, at 24 hpa the mitotic cells were mainly localized in the inter-ray epidermis of the stump. The inhibition of ROS or apoptosis reduced the number of mitotic cells by 50%. In contrast to what it has been described in other models JNK pathway did not seem to be involved in the induction of apoptosis during zebrafish caudal fin regeneration. So it seems that apoptosis and JNK work in parallel downstream of ROS production to induce cell proliferation during regeneration.

Finally the authors checked whether the inhibition of apoptosis or JNK pathway impairs cellular reprogramming or signalling molecules required for blastema growth. Inhibiting the second wave of apoptosis reduced the expression of klf4 and dio3, as NOX inhibition does. However, JNK inhibition did not seem to affect these markers. At the level of signalling pathways, apoptosis inhibition reduced the expression of fgf20, sdf1 and enhanced the expression of igf2b and wnt10a. On the other hand, inhibiting the JNK pathway reduced the expression of sdf1, wnt5b and igf2b. In summary, the authors found out that that the production of ROS during regeneration is important to induce apoptosis and JNK pathway that would work in parallel to promote epidermal cell proliferation and blastema formation during zebrafish caudal fin regeneration.

The Hippo pathway in Macrostomum homeostasis and regeneration

The Hippo signalling pathway has been evolutionary conserved and shown to play a key role in controlling organ size through regulating the balance between cell proliferation and cell death. Also, in mammals the Hippo pathway acts as a tumor suppressor. When the Hippo pathway gets activated the transcriptional coactivator Yorkie (known as Yap in mammals) is repressed. On the other side, when the Hippo pathway is inactive, Yorkie is active. Yorkie induces cell proliferation and inhibits apoptosis and, therefore, its silencing results in a decrease in cell proliferation and increase in cell death.

A recent paper from the laboratory of Eugene Berezikov reports on the function of the Hippo pathway in the flatworm Macrostomum lignano (http://www.ncbi.nlm.nih.gov/pubmed/23495768). Similarly to freshwater planarians, M. lignano is a growing model to study stem cell-based regeneration. All these different flatworms are an excellent model in which to study the role of signalling pathways on stem cell regulation in vivo. In this study, the authors first isolated the homologues in M. lignano of the core components of the Hippo pathway including Hippo (Hpo), Salvador (Sav), Warts (Wts), Mats and Yorkie (Yki/Yap). Whole mount in situ hybridizations showed that in adult animals Hpo, Sav, Wts and Mats show similar expression patterns, being enriched in gonads and also found in neoblasts (pluripotent stem cells) and differentiated tissues. On the other hand, Yap is expressed specifically in the gonads and neoblasts as its expression is completely abrogated after irradiation (a way to eliminate all the neoblasts and dividing cells in flatworms).

Then the authors moved forward to functionally characterize all these genes first in adult homeostatic animals and then during regeneration. In adults, 2 weeks after RNAi treatment to silence Hpo, Sav, Wts and Mats the animals start to develop small outgrowths along the body. After 4-6 weeks there is an increase in the size of these outgrowths and epidermal bulges form throughout the body. Finally, all animals die within 8 weeks of treatment. In contrast, the silencing of Yap results in head regression followed by ventral curling and lysis of the whole animal, a phenotype that resembles those obtained in freshwater planarians when they are depleted of neoblasts. Compared to freshwater planarians M. lignano shows more limited regenerative abilities; however, when amputated behind the head they can regenerate a new posterior part. After Hpo(RNAi) animals regenerate a new posterior part but, remarkably, this new part appears to be larger than in controls. Moreover, the animals also develop bulges around the cutting site and show other morphological aberrations and disrupted allometric scaling. However, cell differentiation does not seem to be impaired in them. All these morphological phenotypes correlate with an increased cell proliferation detected in the blastemas of these animals after silencing Hpo. In contrast the silencing of Yap results in a significant decrease in the number of proliferating cells within the blastema, which blocks the regeneration of a new posterior region. In few days all the animals die.

Finally, the authors used BrdU to label neoblasts after silencing Hpo and Yap in adult homeostatic animals. By day 10 of treatment the number of S-phase cells is significantly higher in Hpo(RNAi) and significantly lower in Yap(RNAi), compared to controls. By day 20 not only the number of S-phase cells was much higher after Hpo(RNAi) but also were found all throughout the body, compared to the more restricted spatial distribution found in controls.

In summary, the results reported in this paper clearly show that the Hippo pathway is conserved in the flatworm M. lignano and, more importantly, that plays a key role in regulating neoblast biology during homeostasis and regeneration. Similarly to mammals the Hippo pathway acts as a tumor suppressor in these flatworms as Hpo(RNAi) animals develop outgrowths and bulges all throughout the body. Also, Yap appears to be required to maintain neoblasts self-renewal, which agrees with the role of Yap in maintaining the pluripotency of mammalian embryonic and induced stem cells. Further studies in M. lignano could help to characterize upstream and downstream elements of this pathway to better understand how the Hippo pathway regulates the size of the regenerating parts by controlling stem cell proliferation, cell death and differentiation.

TNFα links apoptosis to proliferation in zebrafish retinal regeneration

Among vertebrates zebrafish display amazing regenerative capabilities as they can regrow fins, the tail and even the heart. Also, and in contrast to other vertebrates such as mammals, zebrafish can regenerate their retinal cells. Zebrafish retinal regeneration depends upon the activation of Müller glia. After damage in the retina, Müller glia dedifferentiate and re-enter the cell cycle giving rise to a cycling population of multipotent progenitors that will differentiate into the required retinal cell types.

A recent paper from the laboratory of David R. Hyde reports on the role of Tumor Necrosis Factor-Alpha 1 in this process (http://www.ncbi.nlm.nih.gov/pubmed/23575850). Previous studies had shown that following a light-induced photoreceptor cell death there is an increase in the number of cycling Müller glia. This suggested that maybe a factor from the dying cells could activate that Müller glia re-enter the cell cycle. In fact, the injection of homogenates from light-damaged retinas into undamaged eyes is able to increase the number of cycling Müller glia. In contrast, no such increase is observed when injecting homogenates from undamaged retinas. In order to find out proteins present in those homogenates from light-damaged retinas that could activate Müller glia, the authors carried out a comparative proteomic approach. By doing this they identified more than 50 proteins expressed >2-fold in the light-damaged retinal homogenates. One of those proteins was TRAP1 (TNF receptor associated protein 1), which led to the authors check the role of the TNFa signalling pathway in the activation of Müller glia.

Upon light-induced damage the expression of TNFa increases first in apoptotic photoreceptors and later in Müller glia at the time they start to proliferate. Then, the authors carried out several experiments with morpholinos against tnfa delivered at different time points before or after the light-induced damaging of the retinas. A first conclusion of those experiments is that tnfa does not appear to be required for the initial light-induced cell death but is necessary for the activation of Müller glia at later stages of regeneration. Next the authors show that the expression of tnfa in the damaged retina is also necessary for the upregulation of Ascl1a and Stat3 in the Müller glia, which in fact appear also required for a successful regeneration.

Based on previous reports that suggested the existence of different cell subpopulations among Müller glia, the authors propose here a model in which TNFa secreted from apoptotic retinal neurons activates the expression of Ascl1a in PPMg (Primary Proliferating Müller glia). This expression of Ascl1a makes PPMg to re-enter the cell cycle and express Stat3. Then Stat3 induces the expression of tnfa in the PPMg. This new TNFa is secreted and activates the expression of Ascl1 in SPMg (Secondary Proliferating Müller glia), which, in turn, activates the proliferation of those cells. However, the exact relationships between all these different factors within the different types of Müller glia remain to be clearly elucidated.

In summary, this study identifies TNFa signalling produced by apoptotic retinal neurons as the signal that would induce Müller glia proliferation at the initiation of zebrafish retinal regeneration. These results are in agreement with those obtained from other models in which it has been shown that signals coming from apoptotic cells are required for a proper regenerative output (i.e. Hydra). Further studies should try to elucidate the exact role of apoptosis in triggering regeneration in different models and cellular and tissue contexts.

MMPs and planarian homeostasis and regeneration

The extracellular matrix (ECM) does not only provide with a physical or structural support to cells and tissues but also it is a source of signaling and regulatory functions that impact most biological functions. Consequently, regeneration is also a process in which the ECM may obviously play an important role. Thus, for instance, just think on freshwater planarians. During regeneration and homeostasis stem cells proliferate and migrate towards the tissues or structures in which new cells are needed (i.e. the blastema or an old organ). Also, during these processes, the planarians may go through an extensive remodeling of the preexisting tissues. Considering the relevance that the ECM appears to have in regulating cell adhesion, cell migration and stem cell niche, self-renewal or differentiation in other systems, it is surprising how little we know about the role of ECM during planarian regeneration, or even how it changes during the required remodeling associated to regeneration or homeostasis. Now, a recent paper by Isolani and collaborators from the laboratory of Renata Batistoni reports the functional characterization of four matrix metalloproteinases (MMPs) in these animals (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0055649).

Surprisingly, the silencing of two of these MMPs (mmp1, a zinc-dependent proteinase and mt-mmpA, a membrane-anchored proteinase) results in very severe (leading to lethality) phenotypes affecting normal homeostasis in intact planarians, but without apparently impairing regeneration in a significant way. This is true especially for mmp-1, whereas the knockdown of mt-mmpA delays the normal growth and differentiation of the blastema. The silencing of mmp-1 in non-regenerating animals results in tissue disorganization, breaking of the basal lamina and multilayered epidermis. At the cellular level, and compared to controls, the number of proliferating neoblasts appear to increase at 2-3 days of RNAI but then it goes down to the same level as in controls, whereas there is an increase in the expression of post-mitotic markers. On the other hand there is a significant decrease in apoptotic cell death which the authors interpret as mmp-1 being a positive regulator of apoptosis in planarians. In the case of mt-mmpA its silencing in intact non-regenerating animals also leads them to die. However, here, neoblast proliferation is not affected at any time-point, the expression of post-mitotic markers is reduced and authophagy is significantly increased. From BrdU labeling the authors conclude that mt-mmpA may mediate cell migration during homeostasis.

Further experiments are required to better characterize how the silencing of these MMPs alters the ECM as well as any ECM-cell interaction to explain the severe phenotypes observed. It will be also important to determine which cell types (neoblasts or differentiated cells or both) are involved and why these defects do not appear during regeneration. Still, this work may represent a stimulating starting point to characterize the function of ECM during planarian regeneration and homeostasis.

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