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.
Among those structures that can be regenerated by different animals we can find several types of appendages: from amphibian and insect legs to Hydra’s tentacles or arms and siphons from molluscs. Now a recent paper from the laboratory of Dave Ferrier introduces us to the regeneration of the operculum in the polychaete Pomatoceros lamarckii (http://www.ncbi.nlm.nih.gov/pubmed/24799350). As the authors point out there are not many models to study appendage regeneration within the Lophotrocozoans, as compared to other invertebrates (Ecdysozoans) and vertebrates.
P. lamarckii are serpulid polychaetes (annelids) with two types of head appendages capable of regeneration: the radioles (tentacles) for feeding and respiration and the operculum that can close the tube as a defensive strategy. In this study, the authors analyzed how the operculum regenerates at the morphological level as well as taking in account the dynamics of cell proliferation during regeneration. The opercular filament can be divided into two main parts separated by a prominent groove: a basal peduncle and a cup (the operculum). The cup is closed distally by the so-called opercular plate. This plate bears a spine and several prongs.
Upon amputation the first signs of regeneration are the elongation of the stump and the emergence of the new prongs of the spine. Then, by day one a small swelling is observed in the middle of the stump. This swelling enlarges and becomes cup-shaped with an expanding distal plate. Next, the groove that connects the cup with the peduncle is formed. Finally, wing buds develop on the peduncle below this groove. Therefore, it seems that during regeneration the new regions of the opercular filament differentiate following a disto-proximal sequence. In terms of the timing of all these regeneration steps, no significant differences were observed between different sex and size animals.
Next, the authors wanted to investigate the dynamics of cell proliferation during this process. They used two approaches: BrdU labeling and anti-phospho histone H3 immunostaining to detect mitotic cells. One first observation made by the authors was that operculum regeneration proceeds without the formation of a blastema (understood as the formation of an undifferentiated mass of cells at the stump). In fact, during the early stages of regeneration there is very little if any proliferation in the distal plate and spine. Moreover, the lack of BrdU labeled cells in these regions suggests that they do not come from proliferating cells elsewhere. Therefore, it seems that these new distal parts of the opercular filament, including the connective tissue of the cup, regenerate by morphallaxis (remodeling of the pre-existing tissues without cell proliferation). At later stages, the number of proliferative labeled epithelial cells increases in the wall of the new cup and the peduncle and remains high during this stage. At final stages of regeneration, BrdU concentrates in the regenerated wings in the distal peduncle. Remarkably, during the whole process, proliferative cells are uniformly found from the base of the peduncle to the distal cup, with no distinct or preferred proliferation domain.
In summary, it appears that operculum regeneration takes place without blastema formation and through an initial “morphallactic” phase in which the remodeling of the distal stump would give rise to the new distal structures: the spine, plate and the connective tissue of the cup. Later, the regeneration of more proximal regions would depend on cell proliferation, but not at the cut surface but all along the more proximal regenerating regions (from the new cup to peduncle). Also, it is interesting to notice that, morphologically, the new regions appear in a disto-proximal sequence of events.
Future experiments with molecular markers of specific cell types and regions should help to better understand the whole process of operculum regeneration as well as determine the origin of the regenerative cells.
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.
miRNAs are small non-coding RNA molecules that play important functions in transcriptional and post-transcriptional regulation of gene expression. miRNAs have been shown to regulate many important biological processes and be linked to several diseases, cancer and neurogenesis, among others. Moreover, miRNAs have been related to liver and heart regeneration. Now a recent paper from the laboratory of Karen Echeverri reports on the function of miR-125b in spinal cord regeneration in axolotls (http://www.ncbi.nlm.nih.gov/pubmed/24719025).
Whereas in mammals the glial scar inhibits axonal regeneration after spinal cord injury, axolotls have no problems in regenerating their spinal cord. Importantly, no glial scar is detected in axolotls after injury. The rationale behind this study was to concentrate on the environment of the injury site in order to analyze whether intrinsic differences could help to explain the differences between the regenerative capacities shown by axolotls and mammals. That is, the authors tried to identify molecules that promote a regeneration-permissive environment in axolotls.
Previous works by the same research group had focused on analyzing the role of miRNAs in axolotl tail regeneration. Here, they carried out a comparative miRNA profiling analysis at different time points after spinal cord injuries in rats and axolotls. The authors chose 1 and 7 days after complete transection of the spinal cord. Of the approximately 4,000 miRNAs of several vertebrate species included in their array, they found 14 showing significant differences after injury in axolotl versus rat. One of the identified miRNAs was miR-125b, previously related to cancer and stem cell differentiation. Before injury, miR-125b expression in axolotl was concentrated mainly in radial glial cells. In rats, also in an uninjured animal, astrocytes had the highest level of expression; however, in this uninjured situation miR-125b showed 8-fold higher expression in axolotls than in rats. One day after amputation the levels of miR-125b in axolotls decreased about 40%, whereas in rats this decrease was less than 1%. The authors hypothesized that this strong and early downregulation of miR-125b in axolotls could be contributing to creating a permissive environment for regeneration. To test it they analyzed how regeneration proceeded after modulating the levels of miR-125b. The overexpression of miR-125b resulted in an aberrant sprouting of the axons on both sides of the injury together with a reduced growth of the axons through the lesion site by 7 days post-injury, when controls showed normal full regeneration. On the other side, the inhibition of miR-125b in axolotls (up to levels similar to those observed in rats) strongly inhibited also axonal regeneration. Interestingly, this inhibition resulted also in a significant deposition of fibrin that reminded the glial scar found in rats. These results suggested that the levels of miR-125b must to be tightly regulated to allow regeneration in axolotls.
Next, they carried out bioinformatics analyses to try to identify putative target genes regulated by miR-125b. Among them, they identified a homologue to Sema4D, a gene that is upregulated in mice at the wound site after spinal cord injury. In uninjured axolotls Sema4D protein was not detected in glial cells, although it was transiently upregulated at 3 days after injury. On the other side, the inhibition of miR-125b resulted in the upregulation of Sema4D at the injury site; conversely, the overexpression of miR-125b decreased the levels of Sema4D. These results suggested that the regulation of Sema4D levels by miR-125b might play an important role during regeneration. In fact, the overexpression of Sema4D at the injury site inhibited axonal regeneration in axolotl. Then, the authors analyzed the relationship between miR-125b, Sema4D and regeneration in rats. To do that, they modulated in vivo the levels of miR-125b after spinal cord injury. By 7 days post-amputation the levels of miR-125b significantly decreased whereas Sema4D levels increased. The overexpression of miR-125b resulted in a reduced expression of Sema4D, further supporting the results obtained in axolotls. Remarkably, these animals showed improved locomotive abilities compared to controls, reduced glial scar formation and increased number of axons projecting into the scar tissue.
Finally, because the overexpression of miR-125b probably affects the expression of many genes other than Sema4D, they performed microarrays analyses to compare control versus treated animals overexpressing miR-125b. Remarkably, they found that many genes involved in glial-scar formation were downregulated, whereas genes related to neurite outgrowth were upregulated after overexpressing miR-125b.
Taking all the results together, the authors propose a model in which miR-125b would promote a regeneration-permissive environment by downregulating the expression of glial-scar genes and other genes such as Sema4D and, at the same time, inducing positive factor for axonal regrowth. The further characterization of this miRNA-regulated pathway in axolotls and rat can uncover fundamental differences between these two species and help us to understand their different regenerative capabilities.
Regeneration is quite widespread across phylogeny; however, not many species are capable of regenerating a whole animal from a tiny piece of their bodies. And, even in those species, we miss in many of them a deep knowledge of the gene and molecular pathways that control the different events that lead to a successful regeneration. Some positive exceptions are found within Platyhelminthes and Hydra. To understand regeneration from an evolutionary perspective it is necessary to characterize how regeneration occurs at the cellular, molecular and gene levels in as many species as possible from different phyla.
Acoel worms are simple animals belonging to the phylum Acoelomorpha. These animals were classically included within the Platyhelminthes. However, in 1999 some phylogenetic analyses placed them outside the Platyhelminthes. Acoels together with nematodermatids form the phylum Acoelomorpha and for many years have been considered as basal bilaterians. In a new twist, more recent analyses have grouped acoels with Xenoturbella and associated them to Deuterostomates. Therefore, the exact phylogenetic position of acoels is still under discussion. Acoels are capable of whole-body regeneration as previously described for several species (for instance, Isodiametra pulchra) in which it would depend on the presence of adult stem cells.
Now, the laboratory of Peter Reddien has presented a novel model to study whole-body regeneration: the acoel Hofstenia miamia (http://www.ncbi.nlm.nih.gov/pubmed/24768051). These animals are capable of regenerating a new head and tail after transverse amputation. In addition to describing how regeneration takes place in these animals the authors report transcriptomic data corresponding to 16,986 nonredundant gene contigs, which will prove to be an excellent tool for future molecular and comparative analyses. Here, the authors mainly compare regeneration in Hofstenia with what is known in freshwater planarians (Platyhelminhtes). Similarly to what has been described in other acoels regeneration in Hofstenia implies the formation of a regenerative blastema, which appears to be dependent on proliferative stem cells that express the piwi gene marker. Similarly to freshwater planarians most of the mitotic cells concentrate outside the blastema at its base, whereas the blastema would consist of post-mitotic progeny. Also, as in planarians, regeneration appears to proceed through a combination of an epimorphic blastema-dependent stage as well as the remodelling of pre-existing tissues.
The authors also provide evidence that RNAi is efficient in these animals (as it has been proved in other acoels); importantly, in this study, they systematically characterize the expression patterns and silence a large number of genes of the Wnt/b-catenin and BMP signalling pathways to analyse their role during axial re-specification. Remarkably, they found that silencing of b-catenin transform tails into heads whereas the ectopic activation of b-catenin leads to the opposite phenotype: heads are transformed into tails. Therefore, the Wnt/b-catenin pathway plays a conserved role in the re-establishment of AP polarity during regeneration, as it happens in planarians and Hydra. Similarly, their functional analyses with several elements of the BMP pathway revealed its role in DV axial polarity. Thus, the silencing of BMP lead to the ventralization of the dorsal side of these animals.
Finally, the authors carried out some phylogenetic analyses using large-scale transcriptomic data from Hofstenia miamia that, compared to other species, is a slow-rate evolving acoel. From the data obtained the authors support the view that acoels would occupy a basal position among bilaterians, although this should be further corroborated by the analyses of further genomic data from several other species of acoels.
What is important from an evolutionary perspective is the remarkable similarities between how acoels and planarians (separated by more than 500 million years) carry out whole-body regeneration. As the Wnt/b-catenin and BMP pathways are also important for the establishment of axial polarity during the embryonic development of most animals, the fact that they are also required during regeneration in acoels and planarians could be interpreted in two different ways: 1) the last common ancestor of acoels and planarians was able to regenerate using also these pathways, or 2) these pathways were independently co-opted for regeneration in both groups because of their pivotal role in polarity during embryogenesis. On the other side, if the last common ancestor of bilaterians was capable of regenerating it should be possible then to find additional similarities between whole-body regeneration in acoels and planarians, for example. And, certainly, several similarities exist: they have piwi+ proliferative cells with similar distribution, regeneration depends upon those piwi+ cells, regeneration occurs through blastema formation and remodelling of the pre-existing tissues and important patterning genes are expressed in subepidermal domains in the adults of both groups.
In summary, the authors provide here with a new model and tools for further comparative analyses between whole-body regeneration in different species. Those analyses are necessary for a better understanding of regeneration throughout evolution. But we have to keep in mind that as important as to finding similarities is also to uncover differences and analyse them. For example, in Hofstenia the silencing of a gene called notum transforms a head into a tail, as it happens in planarians. However, whereas in planarians notum is expressed in very few cells at the most anterior tip of the head, in Hofstenia this gene is largely expressed throughout the body but completely excluded from the head. Also, in Hofstenia the silencing of admp significantly ventralizes the dorsal side of their body. In planarians, the silencing of admp by itself does not yield an obvious ventralizing phenotype. But, admp silencing enhances the ventralization that occurs in bmp RNAi animals. Therefore, although notum and admp are necessary for the re-establishment of AP and DV polarity, respectively, in both Hofstenia and planarians, the differences described above could be suggesting slight divergences in the fine molecular mechanisms and relationships through which these elements carry out their functions.