The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.     

The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body

It is well known that parts of earthworms can survive if they are cut off. Our aim was to link the regeneration capacity of an earthworm, Eisenia fetida (Oligochaeta, Annelida) with the site of the amputation, so we amputated earthworms at different body segment locations along the length of the body to examine the different survival rates and regeneration lengths of the anterior, posterior, and medial sections.
The greatest survival rates occurred for earthworms with the most body segments remaining after amputation. The anterior regeneration lengths were of two types. The lengths of regeneration of amputated from body segment 6/7 to further down the body posteriorly increased gradually (Type L_I). However, the regeneration lengths of earthworm which were amputated behind the 23rd segment, with less than a quarter of the total segments remaining, did not increase until the blastema and tail bud formation (Type L_II). These treatments were not completely regeneration. There were significant differences in both survival rates and lengths of regeneration lengths between immature earthworms and clitellate adult earthworms during the early stages of regeneration, but not at later stages of regeneration. The immature earthworms had a greater regeneration potential than clitellate adults amputated at the same segment. The survival rates of earthworms were correlated significantly with the number of body segments remaining after amputation, but not with the position of the amputation. The relationships between the survival rates and the numbers of remaining segments could be described by linear regressions. The anterior regeneration lengths were correlated with the position of the amputation, but not with the number of remaining segments; the posterior regeneration lengths, were not correlated with the number of segments remaining nor the amputation position. The anterior regeneration length was not related to the survival rates for all earthworm amputations after 30 days but was related in this way after 60 days.     

Investigating the developmental onset of regenerative potential in the annelid Capitella teleta

An animal's ability to regrow lost tissues or structures can vary greatly during its life cycle. The annelid Capitella teleta exhibits posterior, but not anterior, regeneration as juveniles and adults. In contrast, embryos display only limited replacement of specific tissues. To investigate when during development individuals of C. teleta become capable of regeneration, we assessed the extent to which larvae can regenerate. We hypothesized that larvae exhibit intermediate regeneration potential and demonstrate some features of juvenile regeneration, but do not successfully replace all lost structures. Both anterior and posterior regeneration potential of larvae were evaluated following amputation. We used several methods to analyze wound sites: EdU incorporation to assess cell proliferation; in situ hybridization to assess stem cell and differentiation marker expression; immunohistochemistry and phalloidin staining to determine presence of neurites and muscle fibers, respectively; and observation to assess re-epithelialization and determine regrowth of structures. Wound healing occurred within 6 h of amputation for both anterior and posterior amputations. Cell proliferation at both wound sites was observed for up to 7 days following amputation. In addition, the stem cell marker vasa was expressed at anterior and posterior wound sites. However, growth of new tissue was observed only in posterior amputations. Neurites from the ventral nerve cord were also observed at posterior wound sites. De novo ash expression in the ectoderm of anterior wound sites indicated neuronal cell specification, although the absence of elav expression indicated an inability to progress to neuronal differentiation. In rare instances, cilia and eyes re-formed. Both amputations induced expanded expression of the myogenesis gene MyoD in preexisting tissues. Our results indicate that amputated larvae complete early, but not late, stages of regeneration, which indicates a gradual acquisition of regenerative ability in C. teleta. Furthermore, amputated larvae can metamorphose into burrowing juveniles, including those missing brain and anterior sensory structures. To our knowledge, this is the first study to assess regenerative potential of annelid larvae.     

Variation in Salamander Tail Regeneration Is Associated with Genetic Factors That Determine Tail Morphology

Very little is known about the factors that cause variation in regenerative potential within and between species. Here, we used a genetic approach to identify heritable genetic factors that explain variation in tail regenerative outgrowth. A hybrid ambystomatid salamander (Ambystoma mexicanum x A. andersoni) was crossed to an A. mexicanum and 217 offspring were induced to undergo metamorphosis and attain terrestrial adult morphology using thyroid hormone. Following metamorphosis, each salamander’s tail tip was amputated and allowed to regenerate, and then amputated a second time and allowed to regenerate. Also, DNA was isolated from all individuals and genotypes were determined for 187 molecular markers distributed throughout the genome. The area of tissue that regenerated after the first and second amputations was highly positively correlated across males and females. Males presented wider tails and regenerated more tail tissue during both episodes of regeneration. Approximately 66–68% of the variation in regenerative outgrowth was explained by tail width, while tail length and genetic sex did not explain a significant amount of variation. A small effect QTL was identified as having a sex-independent effect on tail regeneration, but this QTL was only identified for the first episode of regeneration. Several molecular markers significantly affected regenerative outgrowth during both episodes of regeneration, but the effect sizes were small (<4%) and correlated with tail width. The results show that ambysex and minor effect QTL explain variation in adult tail morphology and importantly, tail width. In turn, tail width at the amputation plane largely determines the rate of regenerative outgrowth. Because amputations in this study were made at approximately the same position of the tail, our results resolve an outstanding question in regenerative biology: regenerative outgrowth positively co-varies as a function of tail width at the amputation site.     

Bipolar head regeneration induced by artificial amputation in Enchytraeus japonensis (Annelida, Oligochaeta)

The Enchytraeida Oligochaeta Enchytraeus japonensis propagates asexually by spontaneous autotomy. Normally, each of the 5-10 fragments derived from a single worm regenerates a head anteriorly and a tail posteriorly. Occasionally, however, a head is formed posteriorly in addition to the normal anterior head, resulting in a bipolar worm. This phenomenon prompted us to conduct a series of experiments to clarify how the head and the tail are determined during regeneration in this species. The results showed that (1) bipolar head regeneration occurred only after artificial amputation, and not by spontaneous autotomy, (2) anesthesia before amputation raised the frequency of bipolar head regeneration, and (3) an extraordinarily high proportion of artificially amputated head fragments regenerated posterior heads. Close microscopic observation of body segments showed that each trunk segment has one specific autotomic position, while the head segments anterior to the VIIth segment do not. Only the most posterior segment VII in the head has an autotomic position. Examination just after amputation found that the artificial cutting plane did not correspond to the normal autotomic position in most cases. As time passed, however, the proportion of worms whose cutting planes corresponded to the autotomic position increased. It was suspected that the fragments autotomized after the artificial amputation (corrective autotomy). This post-amputation autotomy was probably inhibited by anesthesia. The rate at which amputated fragments did not autotomize corresponded roughly to the rate of bipolar regeneration. It was hypothesized then that the head regenerated posteriorly if a fragment was not amputated at the precise autotomic position from which it regenerated without succeeding in corrective autotomy.     

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.     

Limb regeneration and apolysis in the tick, Ornithodoros tartakovskyi.

Limb regeneration potential and the apolysis process were investigated in the argasid tick, Ornithodoros tartakovskyi. Developmental instars received single or multiple amputations and were subsequently allowed to undergo single or multiple apolyses. Amputated ticks regenerated complete normal limbs but only after four successive apolyses. Following a single apolysis, the majority of regenerated limbs were essentially miniature duplicates of normal legs but commonly lacked normal chaetotaxy and/or tarsal hump(s). The site of amputation distal to the coxa-trochanter joint, number of limbs removed from an individual, and instar amputated did not consistently influence the extent of regeneration. Coagulation and clot formation were observed.The limbs of the tick apolysed within the old leg hulls. Larvae and nymphs amputated relatively early during the period of apolyses regenerated limbs; late amputations precluded regeneration. The process of apolysis was irreversible and not obviously affected by amputations.     

Patterns of Mitosis and Activation of the Map-Kinase Cascade during Tadpole Tail Regeneration in the Refractory Period of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Development

Patterns of mitotic cells’ distribution and activation of the MAP-kinase cascade during the regeneration of Xenopus laevis tadpole tails were studied before and during the refractory period. It is known that the tadpoles of Xenopus laevis are able to fully restore the full structure of the tail after amputation. However, in the refractory period (stage 45–47), the ability to regenerate is significantly reduced, until its complete absence. The mechanisms of this phenomenon are still poorly understood. We conducted a comparative analysis of the average number of mitotic cells on 0–4 days post amputation in normally regenerating tails and in tails amputated during the refractory period. A significant decrease in the number of proliferating cells throughout the surface of the tail in the refractory period compared with their sharp increase in the blastema area in normally regenerating tadpoles was shown. In addition, we detected activation of the MAP-kinase cascade (dpERK1/2) during normal regeneration and demonstrated its full inhibition during the refractory period. At the same time, in the distal part of the tail amputated in the refractory period, activation of the expression of the regenerative marker gene Fgf20 was not detected. Thus, we can conclude that the blocking of the regenerative capacity in tadpoles during the refractory period is accompanied by a sharp suppression of the mitotic activity of the cells and a misregulation of the activation of the Fgf–MAP-kinase cascade in the tail after amputation.     

Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and fission evolves in regenerative clades

Regeneration, the ability to replace lost body structures, and agametic asexual reproduction, such as fission and budding, are post‐embryonic developmental capabilities widely distributed yet highly variable across animals. Regeneration capabilities vary dramatically both within and across phyla, but the evolution of regeneration ability has rarely been reconstructed in an explicitly phylogenetic context. Agametic reproduction appears strongly associated with high regenerative abilities, and there are also extensive developmental similarities between these two processes, suggesting that the two are evolutionarily related. However, the directionality leading to this relationship remains unclear: while it has been proposed that regeneration precedes asexual reproduction, the reverse hypothesis has also been put forward. Here, we use phylogenetically explicit methods to reconstruct broad patterns of regeneration evolution and formally test these hypotheses about the evolution of fission in the phylum Annelida (segmented worms). We compiled from the literature a large dataset of information on anterior regeneration, posterior regeneration, and fission abilities for 401 species and mapped this information onto a phylogenetic tree based on recent molecular studies. We used Markovian maximum likelihood and Bayesian MCMC methods to evaluate different models for the evolution of regeneration and fission and to estimate the likelihood of each of these traits being present at each node of the tree. Our results strongly support anterior and posterior regeneration ability being present at the basal node of the annelid tree and being lost 18 and 5 times, respectively, but never regained. By contrast, the ability to fission is reconstructed as being absent at the basal node and being gained at least 19 times, with several possible losses. Models assuming independent evolution of regeneration and fission yield significantly lower likelihoods. Our findings suggest that anterior and posterior regeneration are ancestral for Annelida and are consistent with the hypothesis that regenerative ability is required to evolve fission.     

Regeneration of posterior segments and terminal structures in the bearded fireworm,Hermodice carunculata (Annelida: Amphinomidae)

Like many other annelids, bearded fireworms, Hermodice carunculata, are capable of regenerating posterior body segments and terminal structures lost to amputation. Although previous research has examined anterior regeneration in other fireworm species, posterior regenerative ability in fireworms remains poorly studied. As the morphology of the anal lobe (a small, fleshy terminal structure of unknown function) has been used to distinguish East and West Atlantic H. carunculata populations, there is a more imminent need to understand the morphology and organization of tissues in specimens undergoing posterior regeneration, and the timeframe in which significant developmental changes occur. To further investigate this phenomenon, we amputated the posterior segments of living H. carunculata specimens collected from the Gulf of Mexico and monitored posterior regeneration over a 6‐month study period. Although many aspects of posterior regeneration in H. carunculata are consistent with the findings of other annelid regeneration studies, histological analysis revealed that once formed, anal lobe morphology remains relatively unchanged at all stages of posterior regeneration; East Atlantic morphotypes were not observed in the West Atlantic specimens studied here. Additionally, we found that the ventral nerve chord, which is partially responsible for the regeneration of lost body parts in polychaete annelids, terminates within the anal lobe, suggesting that this structure may play a role in the formation of new segments. J. Morphol. 275:1103–1112, 2014. © 2014 Wiley Periodicals, Inc.     

Autophagy is required for zebrafish caudal fin regeneration

Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy – a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging – impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.     

Regeneration of the tail bud in Xenopus embryos.

In an attempt to solve some aspect of the long-standing controversy about the regenerative ability of appendages in vertebrate embryos, the tail bud of Xenopus laevis embryos has beenamputated at stage sranging from St. 26 to St. 32 and its ability to regenerate duringa culture period of 2-3 days has been studied. At amputation stages 26-28,the tail bud consisted only undifferentialted mesoderm and ectoderm, but at stage 32 it had afully differentiated neural tube, a vaculotaed notochord and segmented somites. A total of 137amputations at differnt stages gace consistent results: a tail formed in all the operated larvacand it had normal, well-developed axial tissues in most cases. The relatively few cases with abnormal tail struture were stunted, oedematour larvae with defects in the trunk region as well. It is concluded from these experiments that cells near the original tail budare able to differentiate into tialbud tissues and to replace the amputated regoin, even at these late embryoic stages. The implications of these findings for comparative studies on regeneration in vertebrates are discussed.     

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities¹. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ''trunk'' fragment is cut from an intact worm, a new ''head'' will regenerate anteriorly and a ''tail'' will regenerate posteriorly restoring the original ''head-to-tail'' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as ''neoblasts'' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity^2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters¹⁰, represent an important system for studying evolution of the central nervous system^{11, 12} and have behavioral screening potential^{13, 14.} Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs^{8, 9, 15}. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals^16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.     

TGF-beta signaling is required for multiple processes during Xenopus tail regeneration

Xenopus tadpoles can fully regenerate all major tissue types following tail amputation. TGF-β signaling plays essential roles in growth, repair, specification, and differentiation of tissues throughout development and adulthood. We examined the localization of key components of the TGF-β signaling pathway during regeneration and characterized the effects of loss of TGF-β signaling on multiple regenerative events. Phosphorylated Smad2 (p-Smad2) is initially restricted to the p63+ basal layer of the regenerative epithelium shortly after amputation, and is later found in multiple tissue types in the regeneration bud. TGF-β ligands are also upregulated throughout regeneration. Treatment of amputated tails with SB-431542, a specific and reversible inhibitor of TGF-β signaling, blocks tail regeneration at multiple points. Inhibition of TGF-β signaling immediately following tail amputation reversibly prevents formation of a wound epithelium over the future regeneration bud. Even brief inhibition immediately following amputation is sufficient, however, to irreversibly block the establishment of structures and cell types that characterize regenerating tissue and to prevent the proper activation of BMP and ERK signaling pathways. Inhibition of TGF-β signaling after regeneration has already commenced blocks cell proliferation in the regeneration bud. These data reveal several spatially and temporally distinct roles for TGF-β signaling during regeneration: (1) wound epithelium formation, (2) establishment of regeneration bud structures and signaling cascades, and (3) regulation of cell proliferation.     

Forelimb spike regeneration in Xenopus laevis: Testing for adaptiveness

Experiments were designed to test adaptability of forelimb spike regenerates in Xenopus laevis froglets. The results show that when amputation is at the radius/ulna level, regeneration occurs in 100% of the cases and a single spike of cartilage is the result. The spike regenerates originating from radius/ulna level amputations can be used for feeding and froglet growth is only minimally compromised by the spike. The spike grows in length as the froglet body grows and thus is in homeostasis with the body. The spike develops nuptial pad tissue in reproductively mature males and is occasionally molted, indicating responsiveness to gonadal and thyroid hormones. Finally, and most important, the spike can be used for amplexus and successful mating. In contrast, spikes originating from humerus level amputations were considerably shorter and regeneration from that limb level was less frequent. When amputation was at the body wall regeneration did not occur.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible.     

The influence of the nerve on lower jaw regeneration in the adult newt, Triturus viridescens

The present investigation was undertaken in an attempt to determine the role played by the nerve in the regeneration of the lower jaw of the adult newt, Triturus viridescens. The results indicated that the number of nerve fibers normally available at the amputation surface was very low compared with that of the newt forelimb. Furthermore, denervation of the lower jaw reduced the number of nerve fibers available to an extremely low level and maintained the number at a low level for up to four weeks without intervening redenervations. The regenerative events in the denervated and amputated lower jaws were indistinguishable histologically from those in amputated jaws having normal innervation. This presented an apparent exception to the general rule that regeneration of external body parts is dependent on the nerve. Several possible explanations are proposed by which this apparent exception might be explained. The process following amputation might be an exaggerated form of wound healing and tissue regeneration which can occur in the absence of nerves. The tissues of the lower jaw might be more sensitive to the influence of those nerve fibers present. The nerve fibers themselves might be qualitatively different and thus exert a greater influence on the tissues.