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Reactive Oxygen Species have been implicated in multiple cellular processes. A disturbed redox balance has been associated to cancer and neurodegenerative diseases. On the other hand, redox signalling may also positively modulate some important processes such as immune response, neurological functioning and wound healing. Recently, some papers have shown that ROS play an important role as early triggers of the regenerative response in vertebrates such as Xenopus and zebrafish. In them, ROS seems to play a positive action on cell proliferation, apoptosis and/or the activation of signalling pathways such as Wnt and JNK that ultimately lead to proper blastema formation and regeneration. Now, a recent paper from the laboratory of Karen Smeets reports on the role of ROS in freshwater planarians, which are capable of regenerating a whole animal from a tiny piece of their bodies (http://www.hindawi.com/journals/omcl/2015/392476/abs/).
Firs the authors used carboxy-H2DCFDA to visualize ROS levels during regeneration. Thirty minutes after amputation ROS were detected at the wound regions. Treatment with two types of inhibitors: DPI (a non-specific flavoprotein inhibitor) and APO (an inhibitor of NOX-like enzymes) blocked the induction of ROS at the wound region after amputation. This drug-mediated inhibition of ROS resulted in severe problems of regeneration visualized by the formation of very small blastemas. In contrast, treatment with BSO (an inhibitor glutathione synthesis) and oligomycin A (an inhibitor of ATP synthase) that promote ROS production did not cause any regeneration defect, despite causing an overproduction of ROS at the wound region. In intact, non-regenerating animals treatment with DPI or APO resulted in head regression and lesions ultimately leading to animal death.
DPI treatments at different times before or after amputation indicated that ROS inhibition did not affect wound closure or early stages of regeneration. In contrast to other regeneration models in which ROS inhibition decreases cell proliferation, this does not seem to be the case in planarians. In planarians, regeneration depends on the presence of totipotent stem cells, called neoblasts. No differences in mitotic rates were seen during the first 3 days of regeneration and in intact animals in DPI-treated animals compared to controls. Also, FACS analyses showed no differences in the stem cell population. Next, the authors investigated whether ROS inhibition could be affecting cell differentiation. By using an antibody against SMEDWI-1, a piwi homolog, specifically expressed in neoblasts the authors found that after DPI treatment SMEDWI-1 positive cells accumulated within the blastema compared to controls. In a normal situation the number of SMEDWI-1 positive cells within the blastema decreases over time as those cells fully differentiate into the different cell types. Therefore, an accumulation of SMEDWI-1 within the blastema is interpreted as neoblasts having problems in differentiating. In the case of ROS inhibition this was further supported by the fact that when using markers of early neoblast progeny, those were also significantly reduced. Overall, these results suggested that ROS inhibition might be affecting normal cell differentiation.
Next and to further analyse the possible function of ROS on cell differentiation the authors turned into the central nervous system (CNS). ROS inhibition resulted in a significant reduction of the brain size and in the number of specific neuronal populations as well as defects in the normal pattern of the brain. Remarkably, in some cases the authors found an ectopic expression in posterior blastemas of anterior neural markers. For this reason, the authors studied the polarity determinants Smed-notum and Smed-wnt1 to see whether polarity was affected after ROS inhibition. In anterior blastemas the expression of Smed-notum was not affected during the early stages of regeneration in which polarity is re-established. Interestingly, in few animals Smed-notum was expressed in posterior blastemas by day 1, an expression that was not observed in controls. These results suggest that ROS could be important for posterior identity, although further experiments would be necessary to better understand the role of ROS on the re-establishment of axial identity during planarian regeneration.
In summary, this paper describes the effects that ROS inhibition has on planarians, a model for whole body regeneration. Future experiments should help to clarify the exact role of ROS controlling cell differentiation and patterning in these animals.
An important aspect of stem cell biology is to determine the genes responsible for the maintenance of the different stem cell populations as well as to understand both how they are regulated and how they exert their function. Now, a paper from the laboratories of Ian C. Scott and Bret J. Pearson reports on the identification of the Mediator subunit Med14 as an important regulator of stem cell maintenance in zebrafish and planarians (http://www.ncbi.nlm.nih.gov/pubmed/25772472).
The Mediator complex was first identified in yeasts and its core consists of three modules (head, middle and tail) with an additional kinase module present sometimes. Because in yeast Mediator is located at the promoters of nearly all protein coding genes it has been suggested that it may be part of the general transcription machinery. However, several reports in some plant and animal models do not seem to fully support this model. Interestingly, several subunits of this Mediator complex have been identified as regulators of pluripotency of mouse ESCs. However little is known about the putative role of Mediator on stem cells in vivo.
In this paper the authors characterized the function of the subunit Med14 in stem and progenitor cells and regeneration in two different models: zebrafish and freshwater planarians. First they found that the zebrafish mutant logelei (log) corresponds to mutation of med14. Pleiotropic effects that suggest a developmental arrest characterize the phenotype of this mutant. Accordingly, morpholinos of med14 recapitulate many of these phenotypes, which can be then rescued by the injection of wild-type med14. Analyses of genome-wide transcript levels and mRNA levels did not support a general affectation of transcription in these mutants. In contrast, when the authors focused on stem and progenitor cells and regeneration in the log mutants they observed specific defects. Thus, using different markers they saw that retinal, hematopoietic and gut stem cells were reduced in the log mutant and that med14 seemed to be important in heart progenitor cells. Also, when the tail fin of these animals was amputated it did not regenerate.
In order then to better characterize the putative function of med14 on stem cells and regeneration the authors studied it in planarians, an attractive model in which to study stem cells in vivo. In planarians, as in zebrafish, med14 was ubiquitously expressed including in neoblasts (planarian totipotent stem cells and the only proliferative cells in them). The silencing of med14 by RNAi in intact planarians resulted in a phenotype resembling that obtained after depletion of the neoblasts. That is, the animals curled ventrally, lost their heads and finally lysed. On the other hand, the silencing of med14 also blocked regeneration. After med14 silencing there was a gradual loss of cells expressing the neoblast marker Smedwi-1 (a piwi homologue). There was also a loss of proliferative activity detected with different markers of mitosis and S-phase such as phosphorylated histone H3, histone h2b and pcna. Using a marker of neoblast early progeny the authors found that this cell population was also gradually depleted. In contrast, med14 RNAi did not affect the expression of specific markers for differentiated cell types including neurons, gut cells, muscle, pharynx and eye. Moreover they found a significant increase in apoptosis all throughout the treated animals. These results suggested that med14 silencing affected specifically the neoblasts and their progeny and that transcription in general was not compromised.
In summary, the results presented here indicate that the Mediator complex has an important role in stem cell maintenance in two distant models such as zebrafish and planarians. Further analyses should help determining how the Mediator exerts its function, maybe through establishing the epigenetic landscape essential for pluripotency and stem cell maintenance in these animals.
What are the exact mechanisms that control the re-establishment of axial polarity remains as an important question in many regeneration models. In freshwater planarians several studies in the last years have identified the Wnt/β-catenin signalling pathway as pivotal for the establishment and maintenance of the anteroposterior (AP) axis. Thus, several Wnts and Wnt receptors are expressed in the posterior regions whereas different inhibitors of Wnt signalling are expressed at the anterior end. Consequently, the inhibition of this pathway by silencing either β-catenin or some Wnt genes (such as Wnt1) results in the regeneration of heads instead of tails. Even in intact animals the silencing of these genes transforms the tail region into a head with a proper brain. Conversely, the activation of the Wnt/β-catenin pathway in anterior regions leads to the transformation of those heads into tails. Therefore, it seems quite clear that Wnt/β-catenin is required to specify posterior fates in these animals.
Now, a paper from the laboratory of Kerstin Bartscherer has aimed to identify genes regulated by this pathway (http://www.ncbi.nlm.nih.gov/pubmed/25558068) that could be involved in controlling tissue polarity. In order to identify target genes the authors carried out RNAseq experiments after silencing Smed-β-catenin1 in intact animals. Initially they identified 440 downregulated and 348 upregulated transcripts. Among the downregulated genes they found previously described posterior genes and, conversely, upregulated genes included some known anterior genes. Then, they selected 70 genes downregulated after β-catenin silencing and analysed their expression patterns by in situ hybridization. Of them, 35% displayed differential expression along the AP axis with high expression in the tail. Among the other genes, 21 of them were expressed within or around the digestive system. When they analysed the expression of most of these genes after β-catenin RNAi, they found that their expression was strongly inhibited, validating thus the RNAseq data. Also, they found that many of the upregulated genes after β-catenin RNAi were mainly expressed in anterior regions, as expected.
Next, they selected some of these genes that showed a graded expression along the AP axis and analysed in more detail their expression by FISH. These experiments clearly showed this graded expression, high in the tail and lower towards more anterior regions, strengthening the hypothesis that a β-catenin activity gradient may control gene expression. Recently, it has been described that collagen-positive subepidermal muscle cells express several position control genes (PCGs), which has lead to propose that these muscle cells could somehow provide positional information along the AP axis. Here, the authors show that some of the putative novel targets of β-catenin are also expressed in those posterior muscle cells. In addition, some of these genes were expressed in intestinal muscle cells, which suggests that the gut musculature could be also a source of PCGs. Then, and in order to determine whether planarian neoblasts (totipotent stem cells) differentially expressed the candidate genes sp5 and abdBa along the AP axis they isolated different FACS-sorted cell populations from anterior and posterior regions and quantified their expression in those cell fractions. Interestingly, they found that sp5 and abdBa transcripts were higher in the stem cells from posterior regions compared to anterior neoblasts, suggesting that planarian neoblasts may respond to graded β-catenin activity along the AP axis.
Notum is a secreted Wnt inhibitor that, in planarians, is expressed at the anterior tip of the animal and whose silencing leads to the regeneration of heads instead of tails. It has been proposed that notum regulates early polarity decisions through the inhibition of β-catenin activity at anterior-facing wounds. As the authors reasoned that notum inhibition might induce β-catenin-dependent genes they carried out RNAseq experiments after notum RNAi at 18 hours of regeneration, a stage in which polarity decisions are supposed to occur. By comparing the two sets of genes from the two different RNAseq experiments, they found that 38 of the downregulated genes after β-catenin RNAi were induced after notum silencing. Conversely, 16 of the upregulated genes after β-catenin RNAi were inhibited after notum RNAi. Thus, these 54 genes could represent the genes involved in early polarity decisions during planarian regeneration. Remarkably, 33 out of these 54 genes were also present in a previously generated set of Wnt/β-catenin target genes in zebrafish, which suggests that the common requirement of the Wnt/β-catenin pathway during regeneration in planarians and zebrafish may include a shared set of target genes.
One of the novel candidate target genes identified here was teashirt, a Tsh-related zinc finger protein, a gene that appears to act as modulator of Wnt signalling during Drosophila and Xenopus development. In planarians, teashirt (tsh) was expressed all throughout the central nervous system as well as in a graded pattern in the mesenchyme with highest expression in the tail. Tsh expression was induced upon amputation and, as predicted, it was inhibited after β-catenin RNAi and induced after β-catenin overactivation. Remarkably, a tsh homolog in zebrafish was detected in fin regenerates at 3 days after amputation, being this expression also dependent on the Wnt/β-catenin pathway further supporting an evolutionarily conserved role of Wnt/β-catenin in ensuring correct tissue identity and/or patterning during regeneration. Going back to planarians, the silencing of tsh resulted in two-headed planarians, a phenotype reminiscent of β-catenin and Wnt1 RNAi. These results indicated that tsh would be required to suppress anterior fate at a posterior-facing wound. During posterior regeneration Wnt1 is rapidly induced in the stump. Tsh co-localized with wnt1-positive cells both in intact and regenerating tails. Morevover, tsh was also expressed in neoblasts and that expression seemed to be higher in neoblasts from posterior regions.
In summary, the authors show here that tsh is expressed in wnt-positive cells, probably subepidermal muscle cells, as well as in a subpopulation of neoblasts in both cases in a β-catenin-dependent manner. Tsh could be then a downstream transducer of Wnt signalling important to regulate AP polarity.
Freshwater planarians are among the very few animals capable of fully regenerating a new functional brain from a tiny piece of their bodies. Despite being animals relatively simple from a morphological point of view, several studies have reported the complexity of their central nervous system (CNS). This complexity can be seen by the high degree of molecular compartmentalization based on the expression patterns of many neural-specific genes. In addition, a large number of distinct neuronal populations expressing different neurotransmitters and neuropeptides have been identified.
An important aspect of planarian CNS regeneration is that even though the available tools allow us to determine quite precisely when and where the different neuronal populations regenerate and how the new brain is formed again at the morphological level, much less is known about when this new complex CNS is fully functional again and how the animals recover their normal behaviours. The main reason for this is that few behavioural assays have been established in these animals. So, even in intact non-regenerating planarians there is no much information about which genes might be regulating different behaviours controlled by the planarian sensory system.
Now a recent paper from the laboratory of Kiyokazu Agata describes some genes related to thermosensory signalling in these animals and reports how thermotaxis is re-established during regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25411498). First, they showed that planarians of the species Dugesia japonica displayed normal locomotor activity and morphological shape at a temperature range from 15ºC to 25ºC. At lower temperatures (5-10ºC) their bodies crumpled and lost their motility; above 30ºC they displayed hyperkinesia and became lethargic or even died just after 1 hour. Then, the authors developed a thermotaxis assay based on a method previously used in C. elegans. To put it simple, they created a radial thermal gradient from the centre of a Petri dish towards its edges: the Tª at the centre being 17ºC and at the edges, 25ºC. When the temperature was uniform all throughout the Petri dish planarians moved towards the edges. However, when a thermal gradient was created planarians tended to move towards the centre of the Petri dish and stay there, where the Tª was lower. Decapitated planarians put in a dish with a thermal gradient did not seem to recognize it and moved randomly towards the edges, as they were in a dish with a uniform Tª. In contrast, the small amputated head pieces were able to recognise the gradient and moved towards the centre of the dish, indicating that the head region was necessary and sufficient for thermotaxis in planarians. In all these experiments and assays all the animals displayed the same locomotor activity independently of being intact, headless or head pieces in Petri dishes with a uniform Tª or under a thermal gradient.
Next, the authors investigated when this thermotactic behaviour was recovered during head regeneration. During the first 4 days of regeneration no thermotactic response was seen even though at this stage planarians had already regenerated the eyes and a new small brain. However, a strong recovery of the thermotactic behaviour was observed at day 5 of regeneration. Previous studies had shown that negative phototaxis is also recovered after 5 days of regeneration.
To try to identify genes involved in planarian thermotaxis the authors focussed on the family of Transient Receptor Potential (TRP) ion channels as they have been involved in regulating a variety of sensory systems including thermosensation in different animals. They identified seven genes homologs to TRPs that displayed different expression patterns in planarians. One of them DjTRPMa was expressed in a scattered pattern throughout the body, although there were more cells in the head region that in the rest of the body. Remarkably, the silencing of DjTRPMa by RNAi resulted in a clear defect in thermotaxis. Thus, and contrary to controls, the animals in which DjTRPMa was silenced never rested in the coolest centre area of the Petri dish. Importantly, RNA treatment did not cause any locomotor defect in those animals. That TRP genes are also involved in thermotaxis in planarians was further supported by the results obtained after treating the planarians with AMTB that specifically antagonizes a thermosensitive TRPM family protein in mammals. These animals moved randomly in the Petri dish and did not tend to go the cold central area. Overall, these results suggested that DjTRPMa would be expressed in thermosensory neurons and might be required for thermotaxis.
During regeneration, DjTRPMa-expressing cells appeared de novo by day 2 whereas a normal thermotactic behaviour was not recovered until day 5, suggesting that thermotaxis would depend on something else other than these thermosensory neurons. Then, the authors analysed the effect on thermotaxis of silencing some genes related to different neurotransmitters expressed in specific neuronal populations. Interestingly, the silencing of DjTPH (a marker of serotonergic neurons) resulted in an abnormal thermotactic response (as DjTRPMa did), without disturbing other behaviours as negative phototaxis or proper locomotor activity. Double staining indicated that DjTRPMa and DjTPH were not coexpressed in the same cells. However, neural projections from serotonergic neurons extended towards DjTRPMa-positive cells, pointing out the possibility that serotonergic neurons could somehow transduce to the brain the temperature signals received by DjTRPMa neurons.
In summary, the authors have characterized for the first time a gene that regulates thermotaxis in planarians and analysed how this behaviour is recovered during regeneration. Further studies should help to better characterize the neural circuit that transduces those external signals to the brain and trigger the proper behaviour.
A long-standing question within the field of planarian regeneration relates to the nature of the neoblasts, planarian totipotent stem cells. These cells are the only cells with proliferative capabilities in these animals and are absolutely indispensable for regeneration. Based on morphological criteria (size, shape, nucleus/cytoplasm ratio) about 20-30% of the planarian cells have been considered as neoblasts. However, it has not been clear for many years whether all these cells represent a uniform neoblast population with identical o similar proliferative abilities and potentiality or, alternatively, neoblast comprise a quite heterogeneous cell population respect to these criteria. Recent studies from several laboratories have identified several genes and transcription factors that are expressed in different populations of neoblasts and are required for the differentiation of specific cell types. Based on these data Peter Reddien proposed two models for planarian neoblasts: 1) the naïve model in which neoblasts are a rather homogeneous cell population with the same potentiality and in which fate specification occurs only in the non-dividing neoblast progeny; in contrast, 2) the specialized model predicts that neoblasts are an heterogeneous population containing many different lineage-committed dividing cells.
Based on several recent studies planarian neoblasts seem to follow the specialized model as several cell type-specific genes are co-expressed with Smedwi-1, a planarian piwi homologue, considered as a neoblast marker. Now, a recent study from the laboratory of Peter Reddien further supports the specialized neoblast model (http://www.ncbi.nlm.nih.gov/pubmed/25254346). In this work, the authors first purified by FACS neoblast that were in the S or G2/M phases of the cell cycle (dividing neoblasts) and analysed their expression profiles by RNAseq. Then, they focussed on transcription factors as these proteins regulate cell differentiation. Here, they identified a list of transcription factors that were upregulated in dividing neoblasts during regeneration. To validate these results they next carried out in situ hybridizations on purified dividing neoblasts and all of the transcription factors tested were expressed within these cells although, evidently, with different percentages of positive cells.
They extended then these analyses to other previously characterized transcription factors specific for different cell types. Again, here, they identified a percentage of dividing neoblasts in which these factors were also expressed. Next, they focussed on the planarian nervous system as a target to test the specialized neoblast model as many different neuronal subpopulations have been identified. From the analyses of the transcription factors identified in their RNAseq experiments as well as for the search of conserved factors with conserved functions on the development of the nervous system they found 26 neural transcription factors that were expressed in dividing neoblasts. One of these factors, a klf homologue was coexpressed with cintillo in mechanosensory cells located around the head periphery. Remarkably, klf RNAi lead to the absence of these sensory cells during regeneration, despite these treated animals were capable to normally differentiate many other neuronal cell types. Similarly, a pax3/7 homologue was found to be expressed in the medial region of the brain in cells some of which also expressed a dopamine β-hydroxylase (DBH) gene. Animals in which pax3/7 was silenced by RNAi regenerated a significant reduced number of DBH positive cells. Altogether, these results suggest that these transcription factors would be necessary for the differentiation of specific neural lineages from distinct progenitor neoblast subpopulations.
Importantly, the authors also found that transcription factors associated to specific cell lineages (pharynx, central nervous system, eye, protonephridia or muscle) were not co-expressed in the same dividing neoblasts. These strongly suggest that distinct neoblast populations expressed specific combinations of transcription factors associated to different differentiated cell types.
In summary, this study reports 36 transcription factors expressed in dividing neoblasts from regenerating planarians. These factors are expressed in different cell types and tissues in adult planarians, which suggests that they may specify distinct subpopulations of lineage-committed dividing neoblasts, further supporting the specialized model.
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.
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.
Within the planarian field a hot topic in the last years has been the discussion about which factors control the re-establishment of polarity and whether or not signalling centers functioning as organizers exist during regeneration. Different genes are required to trigger anterior regeneration in terms of providing identity and the proper patterning cues. Among them, wnt1, notum, pbx, prep and follistatin play key roles.
Now, a recent paper from the laboratory of Kerstin Bartscherer report on the central role of the transcription factors foxD (Forkhead) and zicA (zinc finger transcription factor) (http://www.ncbi.nlm.nih.gov/pubmed/24704339). The results presented here complement and expand previous reports on foxD function during planarian regeneration from the laboratories of Peter Reddien (http://www.ncbi.nlm.nih.gov/pubmed/24415944) and Phil Newmark (http://www.ncbi.nlm.nih.gov/pubmed/23297191).
FoxD is expressed at the tip of the head where it co-localizes with other markers of this anterior pole, such as notum and follistatin. Upon its silencing, anterior regeneration was severely impaired as some animals developed small blastemas with cyclopic eyes (eyes fused at the midline) and aberrant small brains, and other worms regenerated extremely small blastemas with no eyes or cephalic ganglia. Next, the authors decided to search for putative downstream targets of foxD by sequencing the transcriptomes of control and foxD(RNAi) regenerating tail pieces. One of the identified candidates was a zinc finger transcription factor that was named Smed-zicA. Remarkably, zicA was also expressed at the anterior tip of the head similarly to foxD, notum and follistatin. In fact, zicA co-localizes with foxD, notum and follistatin-expressing cells. Upon RNAi silencing of zicA, the treated planarians displayed very similar defects on anterior regeneration as those shown after foxD RNAi. To validate that zicA was a target of foxD the authors show how the expression of zicA disappeared after foxD RNAi. However, they also found that foxD expression depended on zicA, which suggests that these two genes may regulate each other’s expression. Also, double silencing of foxD and zicA resulted in more severe phenotypes, further supporting that both genes act together during anterior regeneration.
After either foxD or zicA RNAi the expression of several genes normally expressed at the anterior region was significantly reduced. On the contrary, the expression of genes normally expressed at the posterior pole was unaffected. In planarians notum has been shown to play a key role in the establishment of anterior polarity at very early stages of regeneration. Even though the coalesced notum expression at the anterior tip was inhibited after foxD or zicA RNAi at 3 days of regeneration, the silencing of any of these 2 genes did not affect the early expression of notum. This suggests that foxD and zicA would be required for head regeneration downstream of early polarity determinants. Also, the expression of follistatin at the anterior tip was lost after foxD or zicA RNAi at 3 days of regeneration. Previously, follistatin has been suggested to function together with notum to determine a putative anterior signalling center. In addition, the silencing of foxD or zicA lead to a rapid decrease of the expression of notum and follistatin at the anterior pole of intact non-regenerating animals, just after 3-7 days. After irradiation, however, cells expressing notum and follistatin at the anterior pole were maintained at least until day 8 after treatment. Thus, these results suggest that the loss of notum and follistatin-expressing cells would not be caused by stem cell-based turnover, but would support a role for foxD and zicA in the regulation of the expression of notum and follistatin at those anterior pole cells.
Finally, the authors analyzed whether or not foxD and zicA were expressed in differentiating anterior pole cells, as foxD and zicA-positive cells were lost after irradiation in 3 day-regenerating fragments. They found that foxD and zicA co-localize with the stem cell marker Smedwi-1. But they also co-localize with cells that do not express Smedwi-1 anymore but still are positive for SMEDWI-1 protein. These results indicate that foxD and zicA are expressed in the progenitors of the cells that will give rise to the anterior pole, and somehow required for their differentiation and the formation of this pole. Terminal differentiated cells at the anterior pole express foxD and zicA, whereas progenitor cells closer to the stump co-express these to factors together with SMEDWI-1.
Further experiments are necessary to define the exact relationships between the different genes that appear to be important for the development of this anterior pole (pbx, follistatin, foxD, prep and zicA), as well to see up to which extend this anterior pole works as an organizing signalling center.
Freshwater planarians are well known by their abilities to regenerate a whole animal from a tiny piece of their bodies. This process is driven by pluripotent adult stem cells, the neoblasts (the only cells with mitotic activity). Recently, several studies have reported different specific transcription factors required to commit neoblasts subpopulations into distinct cell lineages. However, it is still unclear how neoblasts are directed into the different lineages that conform, for example, discrete organs. Now, a paper from the laboratory of Alejandro Sánchez-Alvarado describes a novel method to analyse pharynx regeneration in these animals and identifies FoxA as a key factor to regulate a genetic program underlying the regeneration of this organ (http://www.ncbi.nlm.nih.gov/pubmed/24737865).
The planarian pharynx is a peculiar organ because it does not contain neoblasts within it but still depends on neoblasts for its continuous cell renewal and regeneration upon injury or amputation. Previous results had suggested that neoblasts in the body mesenchyme, at the base and around the pharynx, sustain pharynx cell renewal and regeneration by migrating from their original position to the inside of this organ. A remarkable approach used in this paper is that the authors have found a chemical way to selectively remove the whole pharynx from the rest of the animal. Treatment with sodium azide for a short time causes pharynx extrusion and dislodgement. Other organs and systems, including the gut, do not seem to be affected by this treatment. Seven to ten days after this chemical amputation, a new normal pharynx is regenerated in these animals.
After pharynx amputation there is a transient decrease in neoblast mitotic activity, maybe due to the action of sodium azide. In terms of whole-body mitotic activity, this did not significantly change throughout the regenerative process. However, 24 hour after amputation there is a significant increase of proliferation around the wound site. That is, there is a local mitotic peak at 24 hours that then decreases over time. In order to identify transcripts upregulated during this early event of pharynx regeneration the authors isolated plugs of tissues surrounding the pharynx wound site at different time points: 0h, 24h, 48h and 72h. They identified 718 genes that were upregulated at 24h after amputation and cloned 274 of them. Moreover, they cloned 82 genes that were upregulated at 48h and 72h of regeneration, but not at 24h. Some of these genes were validated by in situ, revealing some cases of genes that were not detected in intact uninjured animals but were strongly upregulated at the wound region after amputation.
Next, the authors performed an RNAi screen of the 356 cloned genes. As a read-out to detect possible defects in pharynx regeneration the authors scored the capacity of feeding of those animals. By setting up a threshold of 50% defect in food uptake they found 20 genes. After RNAi of these genes and chemical amputation of their pharynges the authors analysed three features: % of food uptake, pharynx length and mitotic activity in the whole body. According to this, the 20 genes were classified into 3 distinct categories: 1) general regulators of stem cell function as their silencing significantly inhibited neoblast proliferation and pharynx regeneration; 2) specific effectors, whose silencing did not affect neoblast proliferation but regenerated smaller or abnormal pharynges not completely functional; and 3) others, whose silencing did not affect neoblast proliferation or pharynx regeneration but impaired effective food uptake.
One of the genes identified in the category of specific effectors was a homologue of FoxA, a conserved transcription factor required for foregut development in different animals. Planarian FoxA is expressed in different pharyngeal cell types (epithelium, muscle and neurons) and in cells in the mesenchyme surrounding this organ. The authors showed how these mesenchymal cells disappear upon irradiation indicating that they are either neoblasts or early neoblast progenitors. The silencing of FoxA impaired pharynx regeneration upon chemical amputation. Compared to controls, in which after 3 days they had formed a small well-patterned pharynx rudiment, the FoxA RNAi planarians had a disorganized mass of cells. This suggested that FoxA would be required to produce pharyngeal cells and to pattern them properly. In fact, a percentage of cells co-express FoxA and Smedwi-1 (a neoblast specific marker), and this number significantly increases upon pharynx amputation, further supporting that these cells define pharyngeal progenitors. The fact that neoblast proliferation is not affected after FoxA RNAi suggests that FoxA might play a key role in directing neoblast progeny into their differentiation towards pharyngeal cell types.
Finally, the authors analysed the expression of FoxA after silencing the remaining 19 genes identified in their screen defining a molecular pathway driving pharynx regeneration. In summary, this study presents a novel approach to analyse organ regeneration in planarians and identifies FoxA as a key regulator driving pharyngeal cell types differentiation from pluripotent neoblasts.
In this week’s post just let me present to you some of our own recent results on planarian regeneration. After amputation different events must take place in the right order and at the exact time to achieve a successful regeneration. In the case of planarian regeneration one of the first event after wound healing is the establishment of polarity, that is, the animal must know whether to regenerate a head or a tail. Then, once the polarity of the forming blastema is determined other events occur; these include patterning, growth and differentiation of those blastemas. In recent years several genes have been shown to play an important role in the re-establishment of polarity and pole formation in planaria. However, less is known about how for example, a new brain differentiates within an anterior blastema. Now, in a paper from my laboratory, we have characterized a gene that is important for head regeneration in planarians, mainly through its role on the differentiation of the brain primordia (http://www.ncbi.nlm.nih.gov/pubmed/24700819).
In a previous work we had reported that Smed-egfr-3, a homologue of the epidermal growth factor receptor family was important for blastema growth most probably by affecting cell differentiation. Then, we decided to do some DGE analyses to identify putative downstream targets of Smed-egfr-3. One of the isolated genes was Smed-egr-4 a homologue of the early growth response gene family of zinc finger transcription factors.
In situ hybridizations showed that egr-4 was expressed in the CNS, especially the brain, and the mesenchyme in irradiation-insensitive cells (that is, in post mitotic differentiated cells). As expected, the expression of egr-4 was downregulated after egfr-3 RNAi. However, we found out that egr-4 went through two phases of expression, an early expression immediately after amputation or wounding and up to two days, that was independent of egfr-3 and a second phase from the second day of regeneration that depends on egfr-3. The silencing of egr-4 blocked specifically anterior regeneration without affecting the regeneration of posterior regions. In egr-4 RNAi animals, anterior blastemas were very small compared to controls and they failed to regenerate a normal brain or the proper pattern of several other anterior markers. On the other side, the silencing of egr-4 did not seem to largely affect the number of neoblasts (planarian pluripotent stem cells) and although there was a slight decrease in the number of mitotic cells, this did not seem to explain by itself the lack of regeneration. Moreover, other markers suggested that the silencing of egr-4 could be affecting the differentiation of neoblast late progeny.
Recently, other genes have been reported to show a similar phenotype. In those cases, the inhibition of regeneration has been associated to the failure of re-establishing a proper polarity or pole as the expression of polarity determinants is inhibited. Therefore, we wanted to determine whether the impairment of head regeneration observed after egr-4 RNAi was due to defects in the re-establishment of the anterior polarity. However, that did not seem to be the case, after analyzing the expression of several polarity determinants, which suggests that egr-4 might be affecting an event downstream of anterior polarity re-establishment.
As egr-4 was expressed in the mature and differentiated cephalic ganglia we next investigated if egr-4 was required for CNS regeneration. By performing combinatorial RNAi of egr-4 together with an APC homologue we found out that egr-4 appeared to be required for the differentiation of the brain primordia. Given the fact that egr-4 RNAi inhibited anterior but not posterior regeneration and the Wnt/b-catenin pathway mediates the specification of head versus tail regeneration (the silencing of b-catenin1 transforms any blastema into an anterior blastema that will differentiate into a head) we further investigated the relationship between egr-4 and this pathway. Surprisingly double RNAi of egr-4 and b-catenin1 resulted in normal anterior regeneration, which suggested that in a normal situation egr-4 may antagonize b-catenin activity to allow head regeneration. Finally, experiments in which egr-4 was silenced at different time points after amputation and other experiments in which some brain tissues were left after amputation indicated that: (1) the action of egr-4 would be required during the first 2-3 days of regeneration, being dispensable for regeneration after that stage, and (2) the presence of brain tissues was able to rescue the defects observed after the silencing of egr-4.
Overall our results suggest that egr-4 plays a key role in the differentiation of the brain primordia by antagonizing b-catenin function downstream of polarity determinants.
An interesting question raised from these results is that if egr-4 is required for the early differentiation of the cephalic ganglia, how does inhibition of CNS differentiation blocks head regeneration? What we hypothesize is that the brain primordia could send some signal(s) to promote the proliferation, migration and/or differentiation of the neoblasts to allow blastema growth and head regeneration. In the absence of proper brain primordia after egr-4 RNAi, the lack of such putative inducing signal would explain the inhibition of head regeneration. Further experiments are needed to explore this putative relationship between brain differentiation and head regeneration in planarians, which would support the view of an evolutionarily conserved role of the nervous system in animal regeneration.