Many animals are able to regenerate different types of appendages (understood as body wall outgrowths). Among vertebrates, limbs and tails from salamanders and lizards are well-known examples. On the other side, some invertebrates also regenerate appendages such us parapodia, tentacles, opercula, palps and gills. Molluscs comprise a very diverse phylum with an extreme morphological diversity. Up to date, no known representative has been shown to be able to regenerate the whole body. However, several mollusc species can regenerate a variety of structures such as: foot, tentacles, siphon, shell and mantle, and even the head. Cephalopod molluscs are well known for their capacity to regenerate their arms. However, very little quantitative data is available about this process and even less about the cellular and molecular mechanisms involved. In a paper from the laboratory of Jedediah Tressler Nathan J. Tublitz the authors provide a detailed description and quantification of the regeneration process in two species of cuttlefish (http://www.ncbi.nlm.nih.gov/pubmed/23982859). Studying arm regeneration in cephalopods is also important as these structures are as complex as many vertebrates appendages.
It is also obvious that an initial detailed description and quantitative data on the regeneration process in terms of growth rate, behavioural changes, timing of regeneration and functional recovery is necessary prior to tackle the cellular and molecular mechanisms that regulate it, especially in those non-model animals.
First, the authors present the data on Sepia officinalis in which the right 3rd arm was amputated from nine juveniles. In all cases, the amputated arms were regenerated by 39 days, after which the newly formed arms were indistinguishable from the contralateral control arms. They divided the regeneration process in 5 stages: at stage I (days 0-3) the leading edge of the arm appeared smooth with little bleeding. Two days after the amputation the regenerating arm appeared frayed and covered with a mucus-like substance. At this stage only a few new suction cups and no new chromatophores were observed. As a consequence of the amputation the behaviour of the animals was significantly altered as they showed: unbalanced swimming, impaired prey manipulation for ingestion, altered normal body posturing behaviours and lack of any colour change behaviour. Stage II (days 4-15) was characterized by the smooth, slightly hemispherical appearance displayed by the leading edge of the regenerating arm. Growth across the entire width of the arm was symmetrical. New suction cups and chromatophores were seen at this stage. Also, a normal balanced swimming was seen and from day 9 normal food manipulation reappeared. Stage III (days 16-20) began with the appearance of a growth bud on the lateral side of the leading edge, resulting in an asymmetric shape of the regenerating arm. The new arm kept growing at a higher rate that the contralateral control and new suction cups and chromatophores were added at the distal tip. By the end of this stage the regenerating arm was also used for normal body postures. Stage IV (days 21-24) was defined by the emergence of an elongated tip from the growth bud. Suction cup regeneration appeared to be completed by the end of this stage. In the final stage V (days 25-39) the elongated tip took on a tapered appearance. New chromatophores were added until their density in the tip resembled that from control arms. Here, all the checked behaviours, including the brown tip behaviour, were recovered.
In the other species, Sepia pharaonis, arms were also regenerated in 39 days and followed the same 5 stages as in S. officinalis. Based on the location of the new suction cups, chromatophores and the presence of the growth bud and elongated tip, it seemed that new tissue was added directly to the tip of the regenerating arm. Whereas the growth rate of the regenerating arm was quite constant throughout regeneration in S. pharaonis, it varied depending on the stage in S. officinalis.
In summary, in this paper the authors report on the fine description of arm regeneration in two species of cuttlefish at the morphological and behavioural recovery levels. Next step should be to get insights into the cellular and molecular processes governing this regenerative process.
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.
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.
Mammals cannot regenerate their hearts despite that some studies have reported that newborn mice can regenerate the heart apex if it is amputated during their first days of life. Also, in some special pathological or physiological conditions adult cardiomyocytes can be forced to re-enter the cell cycle and/ or dedifferentiate. However, these responses cannot sustain the proper regeneration that should take place after, for example, an acute myocardial infarction. In contrast, zebrafish cardiomyocytes retain their ability to dedifferentiate and re-enter the cell cycle throughout their lives, which explain the amazing capacity of these animals to regenerate a large portion of their hearts. Experimentally, regeneration studies in the zebrafish heart are carried out after either amputation or cryoinjury.
Now a recent paper from the laboratory of Anna Jazwinska reports on the spatial and temporal dynamics of cell proliferation and differentiation during cryoinjury-induced heart regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25557620). The authors first used MCM5 as a marker of the G1/S phase of the cell cycle and phospho histone H3 (PH3) to label mitotic cells in G2/M. Moreover, they used these markers on transgenic zebrafish expressing GFP under the control of the cardiac specific promoter cmlc2. During regeneration the initial fibrotic tissue was progressively replaced with new myocardium, which resulted in the decrease of the infarcted area relative to the entire ventricle. As expected, regeneration was characterized by an increase in cardiomyocytes (CM) proliferation, quite evident by 7 dpci (days post-cryoinjury). During the progression phase at 17 dpci the number of proliferating cells declined and at 30 dpci (termination phase) the mitotic levels had declined up to the levels in uninjured fish. However, at this late stage, the number of MCM5-positive CM still remained significantly higher compared to uninjured animals.
Then, the authors determined the spatial distribution of the dividing CMs relative to the injury border. During the initiation phase (7 dpci) there was a graded distribution of PH3 mitotic cells. Thus, 50% of the PH3 cells were within the first 100 mm adjacent to the cryoinjury border; 20% of them were found between 100 and 200 mm from the border, and the remaining 30% were distributed from the 200 to 1000 mm. Thus, most of the mitotic cells were located at the close vicinity of the wounded area. This graded distribution of mitotic cells was not detected during the progression phase (at 17 dpci) when the PH3 cells were nearly evenly spaced within the entire myocardium. It is important to point out that at this later stage the number of PH3 cells was still significantly higher compared to an uninjured heart. Next the authors performed some BrdU labelling and found out, as expected, that the majority (65%) of CMs located in the regenerated myocardium were BrdU-positive, indicating that they derive from newly generated cells. In the adjacent region (0-100 mm from the regenerated myocardium) about 30% of the CMS were BrdU-positive. Beyond this zone and in all the subregions that the authors checked they found an even percentage of BrdU-positive CMs (about 10%), that again was higher compared to uninjured hearts. Overall, these results suggest that in addition to a local rapid and strong proliferative response to cryoinjury there was also a systemic response all throughout the pre-existing heart.
Finally, the authors analysed the formation of immature CMS during regeneration. To do that they took advantage of an antibody raised against a mammalian neonatal muscle and that binds the N-terminal end of slow b/cardiac myosin heavy chain. In zebrafish this antibody seemed to label embryonic undifferentiated CMs. During regeneration (at 7-10 dpci) the authors found a significant number of CMs positive for this antibody at the injury border within a distance up to 100 mm from the post-infarcted tissue. No labelled cells were found in regions beyond this zone. During the progression stage (17 dpci) these positive CMs lost their initial homogenous alignment along the injury border and intermingled with the regenerated mature cardiac tissue. At 30 dpci almost no labelling was detected. Altogether, the authors concluded that during the initial phase of regeneration, undifferentiated CMS appear at the leading edge of the dedifferentiating adult myocardium and would provide a source of new CMs during regeneration.
In summary, the authors propose that upon cryoinjury a local epimorphic regenerative response is triggered within the 100-200 mm adjacent to the injury border and in which a significant increase in immature and proliferating CMs was detected. This initial response in this blastema-like region would be further reinforced by a systemic compensatory regenerative response within the entire pre-existing myocardium.
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.
In general vertebrates have very limited regenerating capabilities. Among invertebrate chordates however, amphioxus and tunicates show remarkable regenerative abilities. Studying regeneration in these animals is important, as tunicates appear to be the closest living relatives of the vertebrates. Colonial ascidians are capable of whole body regeneration by the activation of stem cells in their vascular system or the epicardium. Solitary ascidians, such as Ciona, show more restricted regenerative capabilities. In addition, it is not clear whether regeneration in these animals depend also on stem cells. Now a recent paper from William R. Jeffery reports on the existence of stem cells in the brachial sac of Ciona, required for regeneration (http://onlinelibrary.wiley.com/doi/10.1002/reg2.26/abstract).
Previous studies had shown that after bisection only basal parts are able to regenerate the distal parts, whereas distal parts are not able to regenerate the missing basal ones. Among the distal parts that can be regenerated researchers have focused mainly on the neural complex and the oral siphon, a muscular tube leading into the pharynx and that contains orange-pigmented sensory organs (OPOs). For both structures regeneration involves the formation of a blastema although the origin of the regenerative cells is not clear.
In this paper, the author focused on the regeneration of the distal oral siphon and its OPOs. First, the author confirmed that only the basal parts were capable of regenerating complete animals. The distal parts did not regenerate any of the proximal (basal) regions and eventually disintegrated. Also, middle body parts were able to regenerate the distal oral siphons with OPOs even in the absence of the basal parts. After the amputation of the oral siphon a blastema containing proliferating cells is formed about 4 days after amputation. Then, he investigated the role of cell proliferation in OPO regeneration. Remarkably, blocking cell division did not affect OPO regeneration. Next, and in order to determine the source of stem cells for distal regeneration two different approaches were used. In a first set of experiments the author labelled dividing cells with EdU for 3, 8, 24 and 48 h after bisection. Control (intact) animals showed EdU labelling in the stomach, intestines and basal stalk. Regenerating animals showed similar levels of EdU labelling in those organs. However, in those regenerating animals a strong EdU labelling was observed from 3 h of regeneration in the transverse vessels of the branchial sac. The labelling was most prominent in the lymph nodes, known to be involved in blood cell renewal. On the other side, the regenerating oral siphon did not show EdU labelling until 48 h of regeneration. Secondly, the location of stem cells was analysed by using alkaline phosphatase (AP) and PIWI markers. In adults, the most intense AP activity was seen in transverse vessels of the branchial sac, where PIWI was also immunodetected. Altogether these results suggest that the transverse vessels of the branchial sac are a potential source of stem cells for distal regeneration.
Next, he carried out several EdU chase experiments to determine the source of the blastema cells. Control and regenerating animals were exposed to EdU for 24 h and then chased without EdU for 5-10 days. Regenerating animals showed an intense labelling in the distal blastema, which was not labelled after 2 days of EdU pulse, which suggests a source of proliferating cells outside the blastema. Then, he transplanted EdU labelled branchial sacs into control hosts that were afterwards bisected at a level leaving the transplanted tissues in the basal part. After 10-15 days of regeneration EdU positive cells were detected in the distally regenerating neural complex and oral siphon. Therefore, it seems that proliferating cells from the branchial sac migrate into the blastema during distal regeneration. Respect to the cells that give rise to the OPOs, experiments with regenerating oral siphons explants suggested that AP labelled stem cells original for the branchial sac would invade the distal areas and differentiate into OPOS during the early stages of regeneration.
The regenerative abilities of Ciona decline with age. So, in a final set of experiments the author wanted to determine whether this decline was related to changes in the stem cells of the branchial sac. He compared the regeneration of young (6 months) and old (12 months) animals. As expected old animals were either unable to regenerate or regenerated partial siphons. Remarkably, the distribution of proliferating cells and the structure of the branchial sacs appeared disorganized in old animals. Moreover, old animals showed very few AP and PIWI labelled cells in the transverse vessels, suggesting that stem cells may be depleted in the branchial sac during aging, being this responsible of their reduced regenerative potential.
In summary, this paper reports on the role of stem cells from the branchial sac in the regeneration of distal structures in Ciona. In these animals the blastema appears to be formed by at least two types of progenitor cells: 1) a subset of branchial sac cells that incorporate EdU very early during regeneration but that they are detected in the blastema after few days (once they migrate there), and 2) another subset of branchial sac cells that migrate to the blastema very early during regeneration and differentiate into the OPOs without undergoing cell division.
I just posted an annual report of this blog prepared by WordPress. I am very happy to see how the number of visits to this site has increased compared to 2013. So thank you very much to all of you that regularly or occasionally read my posts. And also, special thanks to those of you (very few yet) that comment on some of the posts. Unfortunately I couldn’t write any new post the last weeks of December as I got a lot of teaching. But I will be back very soon. So keep ready to read more about regeneration
The WordPress.com stats helper monkeys prepared a 2014 annual report for this blog.
Here’s an excerpt:
The concert hall at the Sydney Opera House holds 2,700 people. This blog was viewed about 13,000 times in 2014. If it were a concert at Sydney Opera House, it would take about 5 sold-out performances for that many people to see it.
Hydra (a diploblastic polyp of the phylum Cnidarian) has been a classical model of regeneration since Abraham Trembley first studied the enormous plasticity of these animals already in the 18th century. Hydra are not constantly renewing their cells but also are capable of regenerating a whole animal from a small piece of their bodies. Remarkably, they are even able to regenerate a well-patterned organism from the re-aggregation of dissociated cells. At the cellular level, Hydra contains three distinct stem cell populations: the ectodermal and endodermal myoepithelial cells are differentiated cells are also stem cells for those specific lineages, respectively, and interstitial stem cells. The interstitial stem cells are multipotent stem cells that give rise to nerve cells, gland cells, nematocytes and gametes. Epithelial stem cells continuously divide in the body column, every 3-4 days and get displaced towards the anterior (tentacles) and posterior (basal disk or foot) tips where they terminally differentiate and progressively get sloughed off. Interstitial stem cells divide also in the body column but at a higher rate, every 24-30 hours and then migrate towards the tips as progenitor cells before their final differentiation.
Now, a paper from the laboratory of Yashoda Ghanekar (http://www.ncbi.nlm.nih.gov/pubmed/25432513) reports the existence of slow-cycling cells within these 3 different compartments of stem cells. In various mammalian stem cell systems, slow-cycling or quiescent cells that do not normally go through division under normal physiological conditions have been described. These cells normally rest in the G0 phase of the cell cycle and divide at a very slow rate or only as a response to injury. Here, the authors report on the presence of slow-cycling cells within Hydra stem cells.
To determine the presence of such slow-cycling cells the authors pulsed Hydra with EdU (a thymidine analog that gets incorporated into the DNA during cell division) for one week to ensure that all cells undergo cell division at least twice and then chased for several weeks in a fresh medium without EdU. Cells that keep dividing will “lose” a detectable EdU signal. On the contrary cells that do not divide any more such as differentiated cells or quiescent stem cells retain the Edu labeling. After one week of pulse, 94-98% of interstitial cell were labeled as well as the 54-80% and the 46-51% of the ectodermal and endodermal epithelial cells, respectively. After four weeks of pulse, these percentages increased to 100% in interstitial cells and more than 90% in epithelial cells. These results indicated that even after 4 weeks few epithelial cells remained undivided. After a long chase (4 weeks) after the EdU pulse, a small but significant number of EdU-positive cells were found in the body column. After one week of pulse and up to ten days of chase around 2.6% of undifferentiated interstitial cells showed complete EdU label. After ten days, only partial labeled interstitial cells were detected (indicating that they were dividing). Ectodermal and endodermal epithelial cells retained the EdU label for much longer. Thus, after a 4 weeks chase, 2.1 and 1.8% of ectodermal and endodermal epithelia cells, respectively, had complete EdU label. Considering the average cell-cycle time of these different lineages, these results suggest that in all three there were cells that did not divide from approximately 8-10 cell cycles after the pulse.
Next, the authors used BrdU (another thymidine analog) and an antibody against mitotic cells to determine that these slow-cycling cells were in fact capable of re-entering cell division. Previous studies have suggested that the extracellular matrix (ECM) could provide a niche for the interstitial stem cells. Interestingly, the authors report here that the percentage of label-retaining interstitial cells in contact with ECM was much higher that that of cells that retained only partial labeling (dividing cells). In other systems quiescent cells are held in G0/G1 phase of the cell cycle. Recently, a study from the laboratory of Brigitte Galliot has reported that interstitial stem cells are paused at G2 phase. After one week of chase for interstitial cells and 3.5 weeks for epithelial cells, most of the cells in these compartments that retained the EdU label were in G2 phase.
Finally, the authors checked the potential contribution of these slow-cycling cells during regeneration. The authors performed midgastric amputation and analyzed head regeneration in animals chased either for one week or 2.5-3.5 weeks. The regenerating tips were cut at 1 and 3 hours of regeneration, macerated and then the authors counted the number of EdU-retaining cells with complete and partial label. As control they used the same body region from animals in chase. The authors found a 50% decrease in the number of cells with complete label at 1h of regeneration and a concomitant increase of cells with partial label, indicating that slow-cycling cells had entered cell division during this time.
In summary, the authors describe here a sub-population of Hydra stem cells that divide infrequently. These slow-cycling cells were present in the 3 stem cell lineages, were capable of re-entering the cell cycle and were activated to divide as a response to amputation during the first hour of regeneration.
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.