regeneration in nature

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Evolution of regeneration in spiralians

One of the on-going debates in the regeneration field concerns how the regenerative capabilities shown by different animal groups have evolved. When considering the animal phylogeny we can see how most phyla contain species capable of regenerating. However, a huge variability exists in: 1) the regeneration power shown by closely related species and 2) the biological level of regeneration as depending on the model they can regenerate only specific cell types, or some tissues and/or organs, or structures and complex parts (for example, a limb) or, finally, the real champions capable of regenerating the whole body.

A recent paper by Alexandra Bely and colleagues discusses about the evolution of regeneration, especially within the spiralians (http://www.ijdb.ehu.es/web/paper/140142ab/regeneration-in-spiralians-evolutionary-patterns-and-developmental-processes). As the authors raise, an important question is whether regeneration variation among bilaterians is the results of regeneration losses, independent gains or a combination of both. That is, was regeneration a feature that was already present in the last common ancestor of all bilaterians and has been lost in some taxonomic groups? or, alternatively, is something that has independently appeared in some groups and not others?

In order to address these questions it is absolutely necessary to gather as much information as possible about the regeneration capabilities of as many animal groups as possible and, more importantly, characterize the cellular and molecular processes that guide regeneration in those animals. Also, it is important to understand why very closely related species can differ significantly in their regenerative capabilities. In this review, the authors focussed on the Spiralia, a large and diverse protostome clade composed of 13 phyla including annelids, molluscs, nemerteans, platyhelminthes and rotifers. Importantly, there is an important variability of regeneration not only between different spiralian phyla but also within them.

Thus, annelids include species that can regenerate every part of the body, including some that can regenerate a whole animal from a single body segment, as well as species totally incapable of regenerating a single segment lost. In general, the capacity to regenerate posterior segments is very broadly distributed within the phylum. In contrast, the ability to regenerate anterior segments is much more variable and, in fact, the failure to regenerate anterior segments has been shown in over a third of the families from which data are available. Nemerteans can undergo growth and degrowth indicating processes of remodelling. Some animals can be maintained starved for over a year shrinking in size but otherwise apparently happy. Among them, regeneration of the proboscis (used for prey capture), tail and head occurs in some groups, and some species can regenerate the whole animal from a tiny body piece. Posterior regeneration is not in general very well documented because the lack of easily scorable structures. Anterior regeneration appears to be very limited within this group although some species of one particular family can regenerate a complete head. However, this seems to be an exception within this phylum, which could imply that it might be a regeneration gain of this particular family.

Among Platyhelminthes, the triclads (planarians) are the best known in terms of their regenerative capabilities. However, it is also true that a number of groups of this phylum have much more limited regenerative capacities. Also, within this phylum posterior regeneration appears to be more widespread than anterior regeneration. Finally, within molluscs we do not have any representative capable of regenerating the whole body. However, different species can regenerate specific structures such as the foot, anterior neural elements, tentacles and even the entire head in some gastropods.

Next, the authors review what is known about the cellular and molecular basis of regeneration within these different phyla. Thus, in annelids after amputation there is a rapid muscle contraction to seal the wound. During the very first stages of wound healing and regeneration, proliferation throughout most of the body seems to be shut down. At the same time there is a large cell migration response towards the wound. After wound healing, cells near the wound start proliferating forming a regenerative blastema. The origin of the regenerative cells within the blastema seems to come from the proliferation of the three tissue layers close to the wound. The role of annelid neoblasts (undifferentiated cells) in regeneration is still under debate. Also, several genes have been shown to be expressed within the blastema including markers of stem cells and germline as well as Hox genes. Interestingly, all these genes expressed within the blastema are also detected during normal growth in the posterior growth zone. This suggests a shared molecular mechanism between regeneration and growth. Finally, regeneration does not uniquely imply the formation of new tissues and structures but also a remodelling of the pre-existing tissues.

In nemerteans, amputation is followed by muscle contraction and wound healing followed by a phase of cell proliferation and the formation of a regenerative blastema, much more evident during anterior regeneration than in posterior regenerates. Unfortunately, the origin of the regenerative cells of the blastema is obscure, and although some old studies pointed to the role of some putative undifferentiated and totipotent cells scattered in the extracellular matrix, more recent studies does not seem to support the existence of such undifferentiated cells. Also, very little is known about the genetic program triggered during regeneration within the blastema cells, except some studies reporting the expression of pax6 and otx in the regenerating central nervous system. Within platyhelminthes, most of the cellular and molecular data of the regenerative process comes from planarians, macrostomids are providing also some interesting data. In planarians, wound healing is followed by the local proliferation of totipotent stem cells (known as neoblasts) closed to the wound that originate a regenerative blastema in which the new structures differentiate. Remodelling of the pre-existing tissues is also necessary to achieve normal body proportions of the regenerated animal. Recently, many papers have reported on different genes and signalling pathways that regulate proper regeneration in planarians. However, much more data should be provided from those taxonomic groups that have either poor regenerative capabilities or for which the cellular and molecular basis of their regenerative capacities are currently unknown. Finally, very little is known about the cellular and molecular processes involved in regeneration in molluscs. A recent report on octopus arm regeneration suggests that a mass of mesenchymal undifferentiated cells would accumulate below the wound forming a highly proliferative blastema.

From all these data and comparative analysis in these spiralian phyla the authors draw four main conclusions: 1) the ability to regenerate the whole body seems to be present in only a subset of representatives of each of these groups. From a phylogenetic perspective numerous increases and/or decreases in regeneration ability have occurred across these phyla. This raises that the possibility that regeneration may not be homologous across them needs to be considered; 2) posterior regeneration appears to be more widespread than anterior regeneration; 3) all phyla include a blastema stage, although the origin of the regenerative cells that form it may be different, and 4) in all these phyla the capacity for continuous growth and degrowth is well documented, suggesting a mechanistic relationship or common set of elements and features between these processes and regeneration.

In summary, how the capacity of regeneration has evolved is also a fascinating field of study that requires much more sampling and data collection for the required comparative analyses.

FoxD, zicA and an anterior organizer in planarians

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.

The role of an early growth response gene in planarian head regeneration

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.

Positional information in planarian muscle

In my las post before the summer break I commented on three papers that had reported how the silencing of a single gene, B-catenin1, was able to rescue head regeneration in three different species of planarians that usually do not regenerate their heads, when amputated post-pharyngeally. Now, in this first post after the holidays I go back to planarians to comment on the recent findings by the laboratory of Peter Reddien on positional information (http://www.ncbi.nlm.nih.gov/pubmed/23954785). As the authors state in their paper, during regeneration, in addition to new cells required for rebuilding the missing structures, these cells must obey very strict instructions in order to be able to form the proper tissues and organs in the appropriate territories. In this sense, no much is known about how positional identities are maintained and re-established during planarian regeneration. Previous studies from the laboratory of Kiyokazu Agata had suggested that such positional information resided in differentiated cells (http://www.ncbi.nlm.nih.gov/pubmed/11319861). Now, the new data from the Reddien’s lab points out that are the muscle cells that would provide such instructions during regeneration.

 First, the authors define the position control genes (PCGs), as genes that (i) display regionalized expression along one or more body axes, and (ii) either their RNAI-mediated silencing results in a patterning defect or encode a protein related to the Wnt, BMP or FGF signalling pathways that are involved in is patterning. These PCGs include: notum, sfrp-1,sfrp-2, several wnts, fz-4, prep, ndk, ndl-3, ndl-4, netrin-2, bmp, admp, ngl-7, ngl-8, nog-1, nog-2 and tolloid. The expression patterns and functions of these genes have been reported in recent years. Interestingly, all these PCGs are expressed in a population of uncharacterized subepidermal differentiated cells. So, the authors wondered whether those cells could represent the source of positional information in planarians.

By doing in situ hybridization with multiple combinations of all the PCGs they found first that many of them co-localized in those subepidermal cells. Next, they found that subepidermal muscle cells that co-expressed collagen, troponin and tropomyosin displayed a similar distribution of those expressing PCGs. Remarkably, every PCG tested was co-expressed with collagen or troponin, suggesting that muscle cells may provide instructive signalling during planarian regeneration. Quantitatively, between 95,7% and 99,8% of all muscle cells analyzed from different body regions co-express PCGs. Although most PCGs are expressed in the subepidermal body wall musculature, others are also expressed in the muscle cells that surround the digestive system or the pharyngeal muscle.

 Finally, the authors analyzed whether the expression of these PCGs was regulated in muscle cells after amputation. Thus, for example, the polarity determinants notum and wnt1 are rapidly induced after amputation in muscle cells. Importantly, this expression occurs in irradiated (neoblast-depleted) animals, suggesting that the existing muscle cells are able to dynamically change the expression of PCGs in them as response to amputation. Moreover, muscle cells are able to re-adjust the profile of the PCGs that express to the one corresponding to the new region along the body axes in which they are placed after amputation.

 Overall the authors propose a model in which changes in the expression of PCGs in muscle cells at the wound regions would influence neoblast cell fate according to their new positions. In the future it would be interesting to test this model by trying to analyze the regenerative capabilities of muscle-deficient planarians (if that is really possible). 

Making heads in regeneration-deficient planarians

One of the many fascinating questions about regeneration is why some animals can regenerate and others not. In this sense, several times in this blog I have pointed out the importance of comparative studies between closely related species with different regenerative capabilities. Last week three independent studies from the laboratories of Phil Newmark (http://www.ncbi.nlm.nih.gov/pubmed/23883929), Jochen Rink (http://www.ncbi.nlm.nih.gov/pubmed/23883932) and Kiyokazu Agata (http://www.ncbi.nlm.nih.gov/pubmed/23883928) published in Nature, reported the recovery of head regrowth in regeneration-deficient planarians after silencing a particular signalling pathway.

Freshwater planarians are among the champions of regeneration as they can regenerate a whole animal (including a complete, functional central nervous system) from a tiny piece of their bodies. However, not all planarian species show the same regenerative competence. One striking observation is that several species can regrow a new head when amputated pre-pharyngeally (in planarians, the pharynx is located in the mid-body region), but those same animals cannot regenerate a head when amputated post-pharyngeally. So, what makes that those tail pieces cannot regenerate a new head even if bearing comparable numbers of stem cells as tail pieces from regeneration-competence species?

In order to address this question the laboratory of Phil Newmark used RNAseq to characterize those transcripts that were differentially expressed between pre-pharyngeal regeneration-proficient and post-pharyngeal regeneration-deficient tissues after amputation, in the species Procotyla fluviatilis. When analyzing the data they realized that many of the transcripts over-represented in the regeneration-deficient tissues corresponded to genes of the Wnt signaling pathway. Previous studies have shown that the Wnt/b-catenin pathway plays a pivotal role in specifying anterior versus posterior identity during planarian regeneration. Thus, the silencing of b-catenin transforms any tail blastema into a head fate. Conversely, the knockdown of an inhibitor of b-catenin (such as APC or axins) transforms any head blastema into tails. These results have been interpreted as b-catenin activity being required to specify posterior identity whereas in the absence of b-catenin anterior identity is specified. Because many Wnt ligands were over-expressed in regeneration-deficient tissues the authors hypothesized that an active Wnt/b-catenin pathway could be the responsible of blocking anterior regeneration in those post-pharyngeal tissues. To test it they simply silenced a b-catenin homologue and, amazingly, a new head regenerated from those regeneration-deficient tail pieces. These results indicate that those regeneration-deficient tissues are competent to express and unfold a head regeneration program but fail at the initial stage of re-establishment of axial polarity. This early step would be necessary to proceed with the subsequent regenerative events (blastema growth and differentiation into a new head). Conversely, an over-activation of the Wnt/b-catenin pathway (through the silencing of the pathway inhibitor APC), lead to the growth of ectopic tails from those regenerating tail pieces.

In the study by Jochen Rink’s lab they worked with the species Dendrocoelum lacteum. Similarly to what it is described for P. fluviatilis, after amputation at the post-pharyngeal level, the resulting tail pieces normally heal the wound and stem cells within it respond to amputation by increasing their proliferation. So, a blastema is initially formed but never grows and differentiates into a new head. In the case of D. lacteum the authors deep sequenced the transcriptomes at 8 eight time points of regeneration (0, 4, 12, 16, 24, 48, 72 and 120 hours) from pre-pharyngeal regeneration-competent tissues and post-pharyngeal regeneration-deficient tissues. In both cases, amputation lead to the normal upregulation of a collection of previously reported wound-response genes as well as a similar stem cell activation response. However, the authors saw how tail pieces failed to activate a set of head-specific genes while at the same time maintained the expression of tail markers. This was in sharp contrast to what happens in pre-pharyngeal regenerating tissues in which head markers were activated and the expression of tail markers was downregulated. Because what it is known about the Wnt/b-catenin pathway the authors speculated that the differences between pre-pharyngeal and post-pharyngeal regenerating pieces could be due either from inappropriate high levels of Wnt signaling and/or from insufficient Wnt inhibitory capacity. Therefore, they silenced b-catenin in tail pieces and, as a result, these were able then to regenerate a new head. Conversely, the silencing of APC gave rise to the regeneration of ectopic tails.

Finally, the work by Kiyokazu Agata and Yoshihiko Umesono followed a complete different strategy in the planarian Phagocata kawakatsui but also showed how the silencing of b-catenin rescues head regeneration from regeneration-deficient tail pieces.

In summary, the three studies show how just by simply modulating the Wnt/b-catenin signalling pathway you can transform a regeneration-deficient tissue into a regeneration-competent tissue capable then of re-growing a new head with a fully functional and complex brain. Further studies should determine why in these species the post-pharyngeal pieces, upon amputation, fail to modulate the Wnt/b-catenin pathway to re-specify proper polarity and subsequently trigger the head-regeneration program. These terribly exciting results may help us to understand the mechanisms responsible for the loss of regeneration capacities through evolution as well as provide insights into how to promote regrowth in regeneration-deficient models.

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.

Regeneration of the symbiotic flatworm Paracatenula galateia

Several times in this blog I have pointed out the importance to extend our regenerative studies to more different species in order to have a broader view of this fascinating process as well as for a more comparative approach. Platyhelminthes are amazing animals not only because many of them show very high regenerative capabilities but also because these are based upon the presence of adult pluripotent stem cells. Among Platyhelminthes, model species include the freshwater planarians Schmidtea mediterranea and Dugesia japonica as well as the marine flatworm Macrostomum lignano.

A report from Ulrich Dirks and Jörg A. Ott has started to characterize the proliferative response associated to the regenerative process in the Catenulida, Paratenula galateia (http://www.ncbi.nlm.nih.gov/pubmed/22729484). Catenulida represent the most basally branching group of the Platyhelminthes. P. galateia is an interesting free-living animal with no mouth and gut, that reproduces asexually by paratomy and that harbors intracellular bacterial symbionts. These animals can be divided into an anterior rostrum and the trophosome region where the symbionts are found. In the rostrum these animals have a brain from which rostral nerves extend up to the most anterior tip. Two main longitudinal nerves extend from the rostrum towards the posterior regions of the animal. S-phase cells localize mostly throughout the trophosome up to behind the brain, so not dividing cells are seen in the most anterior part of the rostrum. When amputated behind the brain, the trophosome is able to regenerate a new rostrum; however, the rostrum is not able to regenerate the trophosome.

The authors analyse the dynamics of neoblast proliferation by combining EdU and BrdU pulses during rostrum regeneration. By 48h of regeneration the wound is closed by the constriction of circular muscles and the flattening of epidermal cells. At this stage EdU and BrdU positive cells are evenly distributed in the whole trophosome fragment. On the other side, stainings with an anti-serotonin antibody show the truncated longitudinal cords in the wound area. After 5 days of regeneration there is strong accumulation of proliferative cells within the forming blastema.  Interestingly, an accumulation of proliferating cells is also found along the longitudinal nerve cords. At this stage, however, the amputated nerve cords appear still truncated and not extending into the blastema. The rest of the body shows an even distribution of low density of proliferating cells. Then, around 7 days of regeneration the new tip of the rostrum starts growing and a strong accumulation of proliferating cells is still observed in the blastema. Also, a prominent commissure appears at the anterior end of the nerve cords and some neuronal processes extend anteriorly, possibly corresponding to the regenerating rostral nerves. Finally, regeneration appears to be practically completed by day 11 after amputation. On the other side, all the rostrum fragments die after a couple of weeks without any sign of posterior regeneration.

Thus, this study represents a first step in trying to determine the proliferative dynamics of the neoblasts during regeneration in this Catenulida. However, and as the authors state, a more detailed time course of the regenerating process in terms of the proliferative response of the neoblasts is needed. Also, it will be interesting to further investigate how these neoblasts within the blastema exit the cell cycle and differentiate into the different cell types. Finally, it would be also interesting to study whether the failure to regenerate posterior regions from a rostrum fragment is caused by the low number of neoblasts present in the rostrum or the fact that the rostrum lacks enough symbionts for its nutrition. Maybe it would be interesting to analyze, in case the authors have not done it yet, what happens when the amputation is done not behind the brain but through the middle region of the trophosome. Would then the anterior fragment bearing the rostrum and part of the trophosome be able to regenerate the posterior end?

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