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Among the few animals that can regenerate a whole brain, freshwater planarians and some annelids are well-studied models, although our knowledge on how this process is achieved at the molecular level is much more limited in annelids. Now, a recent paper from the laboratory of Barbara Plytycz reports on the role that the immune system may play in the regeneration of the brain in the earthworm Dendrobaena veneta (http://www.ncbi.nlm.nih.gov/pubmed/25863277). In this species, the brain is constituted by two hemiganglia, which are connected to the subpharyngeal ganglion and the ventral nerve cord.
In their experimental set up to remove the brain of the worms the authors used two different strategies. In one of them the worms were temporarily immobilized by an electric shock whereas others were anesthetized with Prilocaine. These two methods had different impacts on the immune system of the treated animals. In the case of the electroshock it induced the extrusion of the coleomic fluid within which free-floating coelomocytes and soluble factors involved in the immune response are present. However, Prilocaine resulted in a minimum loss of coelomic fluid, thus preserving the components of the immune system. The authors then compared brain regeneration in these two different scenarios. At the cellular and histological levels the D. veneta brain is characterized by: i) a cellular layer of neurons and glial cells of heterogeneous cell size and larger cells with eosinophilic, basophilic and polychromatophilic staining properties; ii) a well-structured neuropile; iii) very few diving cells in the neural tissues but some within the perineural sheet; and iv) a perineural sheet rich in capillaries.
When using these features to characterize the regenerated brains 4 weeks after surgery the authors found out that the worms that went through electric shock (coelomocyte-extruded worms) regenerated smaller and less organized brains compared to those that went through the Prilocaine treatment. Thus, these brains had a thinner cellular layer, a seemingly random organization of nerve fibers in the neuropile, few mitotic cells in the regenerating brain and its surroundings and very poor presence of capillaries. In contrast, the regenerated brains of the Prilocaine-treated animals displayed many histological features very similar to those from the controls. Moreover, a significant number of mitotic cells were found within the regenerated brain and its surroundings.
As brain function has been also linked to regulation of reproduction in these animals the authors next checked whether this faster and more successful brain regeneration in this last group of animals was also linked to a faster recovery of reproduction. Measures of the number of cocoons produced at several times after brain removal as well as number of hatchlings from those cocoons indicated that the reproductive output after brain removal was restored faster in worms with a relatively complete coelomocyte complement (Prilocaine treatment) than in coelomocye-depleted animals (electro-shock).
In summary, the results presented here further support the notion that coleomocytes play an important role during brain regeneration in earthworms as well as reproduction in them is controlled by neurosecretions. Further studies should try to determine the exact role that a putative factor(s) from the coelomic fluid has on regeneration either by promoting the proliferation, survival or differentiation of the regenerated brain cells.
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.
Among invertebrates some annelid species show also remarkable regenerative abilities. Within this group of animals, however, it is not rare to find species that can regenerate posterior regions but not anteriorly. Also, regeneration has not been extensively studied in all taxa. Now, a recent paper from the laboratory of Michael Weidhase and Conrad Helm (http://www.ncbi.nlm.nih.gov/pubmed/25392962) describes for the first time the process of anterior regeneration in the annelid Cirratulus cirratus, focussing mainly in the musculature and central nervous system (CNS). The authors used phalloidin to label the muscle and antibodies against serotonin and FMRFamide to observe the regenerating CNS.
As for the body wall musculature, it is mainly composed of an outer layer of circular fibers and an inner longitudinal muscle layer. The dorsal longitudinal muscles form a plate that covers the whole dorsal body. Ventrally, there is a medial and two ventro-lateral strands of longitudinal muscle. On the other hand, the CNS is a rope-ladder-like system. The ventral nerve cord consists of two main strands and each of them exhibits one ganglion per body segment. From these ganglia there are three major nerves that extend laterally. At the first chaetigerous segment both strands of the ventral nerve cord separate from each other to form the circumesophageal connective surrounding the mouth opening. This connective splits into a dorsal and a ventral root. The brain is located at the anterior end of the circumesophageal connective. Anti-serotonin immunostaining revealed two brain commissures: a stronger stained anterior commissure connected to the ventral root of the circumesophageal connective and a slighter stained posterior commissure connected with the dorsal root.
After amputation, the wound was closed and a blastema was formed within the first week. Then this blastema elongated anteriorly and about 10 days after decapitation the new mouth opening was evident. By the end of the second week the first outgrowing tentacles appeared. The whole regeneration process took place in about 1 month, although the regenerated head showed some differences with the original respect to the number and length of tentacles and branchiae.
By day 6 of regeneration a blastema was formed and contained outgrowths from the pre-existing longitudinal muscle fibers. From that time the longitudinal musculature kept growing and organized into muscular strands. By day 10 the ventral longitudinal strands reached their most anterior position, surrounding the regenerated musculature of the new mouth opening. At this stage the first circular muscle fibers appeared.
Concerning the nervous system, by day 6 after decapitation, a structure represented by three nerve loops was visible inside the blastema, one median loop and two laterals. At this stage the median loop was folded backwards and was connected to the inner bundles of both strands of the ventral nerve cord. On the other hand, the lateral loops were already oriented anteriorly and were connected to the outer bundles of the ventral nerve cord. At day 8 of regeneration the median loop was also oriented anteriorly. This tripartite loop-like structure has not been described in other regenerating annelids. Whereas the authors favour that the median loop will originate the ventral root of the regenerated circumesophageal connective (visible by day 12), they did not obtain clear data about the transition from the lateral loops to the dorsal root. Finally, the brain commissures were visible by day 14 of regeneration. At this stage, the regenerated nervous system elongated together with the blastema; however, distinct ganglia and lateral processes in the new nerve cords were still missing by day 18. Later, the first signs of body segmentation and differentiation of distinct ganglia and lateral nerves were observed.
In summary, this paper describes for the first time anterior regeneration in the annelid Cirratulus cirratus. Further studies are necessary to describe in more detail this process as well as to determine the origin of the regenerative cells that form the blastema and the new muscle fibers and central nervous system, among all the other anterior tissues.
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.
Hox genes code for transcription factors that play an essential role in the regionalization of the anterior-posterior axis in animals. Thus, they function by providing specific identities to the different segments of the body plan, which translates in the differentiation of the correct structures in each segment. Well-known and astonishing examples of homeotic transformations include those that transform Drosophila antennas into legs or flies with two pairs of wings. Hox genes have been highly conserved through evolution and, for example, Hox genes from flies can replace the function of their vertebrate orthologs.
The function of Hox genes has been (and still is) largely studied during the embryonic development of a large number of invertebrate and vertebrate species. During regeneration, specially in those cases in which large structures or body regions need to be re-grown, it is easy to imagine that Hox genes should have an important function in the re-establishment of the positional identity within the regenerate. However, and in contrast to the observations that conserved signalling pathways such us the BMP, Wnt/beta-catenin, Hedgehog and FGFs play important functions during regeneration, many less functional studies have been reported on the role of Hox genes during regeneration. Thus, whereas several families of genes containing an homeobox domain (pbx, prox, pitx, six, Rx, msx,…) have been functionally characterized during regeneration in planarians, tunicates or amphibians, most of the studies on Hox genes and regeneration focus on their changes in expression during this process. Some of those studies were important for example to show how Hoxc10L is not expressed during axolotl forelimb development but it is upregulated during regeneration, pointing to a “regeneration-specific” expression of this gene (http://www.ncbi.nlm.nih.gov/pubmed/11150241). Also, few years ago it was reported that Hoxc13 orthologs are important for zebrafish tail regeneration where are required for blastema cells proliferation and growth (http://www.ncbi.nlm.nih.gov/pubmed/17437127).
Now, a recent paper from the laboratory of Elena Nokikova and Milana Kulakova reports on the expression of 10 Hox genes during regeneration in the polychaete Alitta (Nereis) virens (http://www.ncbi.nlm.nih.gov/pubmed/23638687). During postlarval development the Hox genes are mainly expressed in a posterior-to-anterior gradient mode. These annelids can regenerate their posterior body end. After amputation the terminal pygidial structures and prepygidial growth zone (GZ) form first and then the new segments appear sequentially. The authors divided the Hox genes into four groups depending on their changes in expression during regeneration. Three genes (early genes: Nvi-Lox5, Nvi-Lox2 and Nvi-Post2) were rapidly upregulated in the nervous system near to the amputation plane by 4 hpa (hours post amputation). Then, Nvi-Hox5 and Nvi-Hox7 (middle genes) expression patterns in the nervous system were reorganized by 10 and 18 hpa, respectively, before active proliferation in the GZ started. Next, two Hox genes, Nvi-Hox2 and Nvi-Hox3 (middle genes), that are not expressed in a graded manner but specifically found in the GZ were upregulated de novo by 10 hpa. Nvi-Hox2 first appeared in two bilateral domains at the amputation plane. By 2 dpa (days post amputation) this gene was expressed in the mesoderm and ectoderm of the area between the forming pygidium and the last body segment. By 7 dpa it was detected in the mesoderm of the GZ and the mesoderm and ectoderm of the newly formed segments. Nivi-Hox3 was also first seen by 10 hpa and as regeneration proceeds got restricted to the ectoderm of the GZ. Finally, there are three genes (late genes: Nvi-Hox1, Nvi-Hox4 and Nvi-Lox4) whose expression patterns changed at late stages of regeneration once proliferation and organogenesis were under their way.
Based on all these expression patterns and dynamics the authors divided the regeneration process in two phases: during the first 48 hpa the expression patterns were reorganized inside the new body boundaries. The second phase (that overlaps with the first one) started around 24 hpa when the blastema was formed. Most of the Hox genes were highly expressed within the blastema and the rudiment of the terminal structures that were evident by 3 dpa.
Because during postlarval development Hox genes are expressed in a anterior-to-posterior gradient in segments that are morphologically similar, the authors suggest that Hox genes at this stage provide the positional information needed to determine the position of body parts. Thus, after amputation the expression of most of the Hox genes of the early and middle groups is reorganized so the last body segments adjacent to the amputation plane acquire the Hox pattern typical of the posterior body end. Remarkably the blastema seems to be formed after the Hox genes have been reorganized to the new body proportions.
Future functional experiments should help to determine the exact role of the Hox genes during not only the re-patterning of the body during polychaete regeneration but also in other models such as planarians or amphibians.
Different mechanisms are used to specify new segments during, for example, somitogenesis in vertebrates and Drosophila body segmentation. Other segmented animals as some annelids keep adding segments as they grow. In a recent paper the laboratory of Shigeo Hayashi describes how new segments are added during the regeneration of the tail in the polychaete Perinereis nuntia (http://www.ncbi.nlm.nih.gov/pubmed/23608458). In many models the new posterior segments are added from a segment-addition zone.
In this study the authors work on the species P. nuntia that, in laboratory conditions, adds a new posterior segment every 4 days. This new segment is added between the last posterior segment and the terminal end, called pygidium. During tail regeneration, a new pygidium is formed and then new segments are added initially at a rate of 1 segment per day. First, the authors show how during normal growth of segments it appears a single row of cells with high PCNA (proliferating nuclear antigen) expression at the border between the last segment and the pygidium. Because PCNA expression is high in late G1-S phase these observations suggest that this single row of cells are in synchronous G1-S phase and has been named by the authors as “zone of cell-cycle synchronization” (ZCS). In addition, and in contrast to the quite random orientation of the mitoses in more anterior regions of the segment, the cells in the ZCS divide along the AP axis with the division axis almost perpendicular to the segmental furrow. The authors also show how changes in PCNA staining are related to changes in chromatin structure based on labelling of the histone modifications H3K9me2 and H3K9, K14ac. Blastema cells are characterized by high H3K9me2 and proliferation rate, whereas when cells differentiate exit the cell cycle and display high level of acetylated histone H3.
Next, the authors follow the regeneration of the new segments by analysing the expression of wingless (wg) and hedgehog (hh). These two genes are expressed in rows of cells at the anterior and posterior side of the segmental furrow, respectively. These cells also overlap with the ZCS of cells with high PCNA expression. Remarkably, the expression of tcf, a nuclear effector of Wg signalling is expressed in a segmental pattern peaking at the hh stripe and decreases in a posterior gradient, suggesting an important role of Wg signalling during segmentation. In fact, treatment with LiCl, which mimics overexpression of Wg signalling produces various regeneration defects. Thus, the row of cells with high PCNA expression at the posterior border of the newly formed segment is disorganized and the animals show a decrease in segment number and segments wider than in controls.
From these results the author suggest a model in which the last row of the most posterior segment serves as the source of Wg that coordinates cell-cycle entry and recruitment of anterior pygidium cells that will initiate the formation of a new segment in the ZCS. Thus, and in contrast to other models, it appears that in P. nuntia segmentation is initiated by cell cycle synchronization and row-by-row addition of cells to the posterior end of the new segment.
Several times in posts and comments in this blog we have discussed the necessity of more comparisons between not only different un-related (phylogenetically) regenerative models but also between closely related species with different regenerative potentials. In a paper published last year, Maroko Myohara tells us about the role that neoblasts may have during regeneration in annelids (http://www.ncbi.nlm.nih.gov/pubmed/22615975). Although these days the term “neoblast” is mainly associated to the somatic pluripotent stem cells found in planarians (see some recent posts and comments), the truth is that, as the authors remind to us, the term “neoblast” was coined by Randolph in the late 19th century to refer to some cells that participate during regeneration in the oligochaete annelid Lumbricus. In fact, planarian and annelid neoblasts are not so similar, neither at the morphological level nor in terms of stem cell properties. In planarians, neoblasts are small cells, widely distributed in the animal, representing about 20-25% of total cells and the only cells that can divide. Moreover, at least a proportion of them, behave as real pluripotent stem cells in terms of self-renewal and pluripotency (all this, despite some recent proposed needs to re-define planarian neoblasts). On the other hand, neoblasts in annelids are large cells, relatively few in numbers, appear to occupy rather specific locations in the body (at the intersegmental septa along the nerve cords) and are more prominent in those species that reproduce asexually. In terms of their differentiation potential, annelid neoblasts appear to have a role in the regeneration of mesodermal tissues, whereas ectodermal and endodermal tissues regenerate from the dedifferentiation and proliferation of cells from the same layers. Thus, in annelids, neoblasts are not the only cells that divide. More importantly, self-renewal and pluripotency have not yet determined experimentally in annelid neoblasts.
In this paper the author tells us about two species of the same genus of oligochaetes that show very different regenerative capabilities, Enchytraeus japonensis and Enchytraeus buchlolzi. Whereas E. japonensis worms have neoblasts in each of the body segments (except the 7 head segments and the first trunk segment), E. buchlolzi lack neoblasts throughout their body. Another important difference is that E. japonensis reproduces asexually (also sexually under some circumstances) and E. buchlolzi undergoes exclusively sexual reproduction. In terms of regeneration, E. japonensis worms amputated at any level along the AP axis of the trunk (neoblast-bearing segments) regenerate anteriorly normal heads and posteriorly normal tails. One remarkable feature of anterior regeneration from trunk pieces is that regardless of the level of amputation E. japonensis regenerates only the seven head-specific segments (no matter how many anterior segments were missing). That suggests that after amputation the most anterior facing trunk piece must adopt a positional identity value equivalent to the original 8th segment, regardless of its position along the AP axis before amputation. On the other hand, when worms are amputated through the head region, only the segments missed are regenerated. But respect to posterior regeneration from head fragments, these typically regenerate head segments instead of tails, producing dicephalic worms (that is, there is a reversal of polarity in the regenerated part). So, head fragments that lack neoblasts are also able to regenerate, as in fact it has been also shown for other annelids that lack neoblasts.
What happens in E. buchlolzi? In anterior regeneration, after amputation through the head or anterior trunk (8th-14th segment) regions, worms regenerate few head segments (never the seven head-specific segments), which result in hypomeric heads. Also, some examples of reversal of polarity are found when the amputation is made in the posterior trunk region as sometimes tails instead of heads are regenerated. During posterior regeneration, trunk pieces normally regenerate proper tails but head pieces are incapable of regenerating any tissue and in fact die in few days.
In summary this paper shows how annelids display different regenerative strategies and capabilities. Some of them can regenerate despite lacking neoblasts; others, have neoblasts and use them to regenerate although regeneration is also possible from regions that lack neoblasts (for example from head pieces in E. japonensis); even in the same animals different tissues regenerate either from neoblasts or dedifferentiation of mature tissues (mesodermal derivatives from neoblasts and ectodermal and endodermal from the same layers). In addition, this paper strengths the known relationship between regenerative potential and sexual vs asexual reproduction. In the future, it will be interesting to characterize better, among others, how annelid neoblasts behave during regeneration, determine their potentiality and self-renewal capacity and also, what are the factors responsible for the different regenerative responses observed depending on the level of amputation along the AP axis.