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.     

Morphological,cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.     

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.     

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.     

Effects of maternal investment on larvae and juveniles of the annelid Capitella teleta determined by experimental reduction of embryo energy content

Experimental manipulations of the energy content of marine invertebrate embryos have been useful in testing key assumptions of life history theory, especially those concerning relationships between egg size, length of the planktonic period, and juvenile size and quality. However, methods for such “allometric engineering” experiments have been available for only a limited set of taxa (those with regulative early development, e.g., cnidarians and echinoderms). Here, we describe a method for the reduction of embryo energy content in the spirally cleaving embryos of a marine annelid, Capitella teleta, by targeted deletion of endodermal precursor cells. Embryos of C. teleta in which up to three cells (the macromeres 3A, 3B, and 3C) were deleted formed morphologically normal lecithotrophic larvae that were much smaller than larvae developing from control embryos. Experimental larvae metamorphosed at high rates, forming juveniles that were smaller than control juveniles. Juveniles derived from treated embryos had functional midguts, ingested and digested food, and grew into sexually mature adults. These results are consistent with those from previous allometric engineering studies of echinoid echinoderms, which suggest that in facultatively planktotrophic or lecithotrophic species, little maternally derived energy is used for construction of the larval body; instead, the majority is allocated to the formation of a large, high‐quality juvenile. Cleavage programs are highly conserved among divergent spiralian taxa (e.g., molluscs, nemerteans, and platyhelminths), so this method will likely be applicable to a diverse set of embryos. Similar experiments carried out in these diverse taxa will be extremely useful for evaluating inferences on relationships between egg size, length of the planktonic period, and juvenile size and quality previously based only on experiments on echinoid echinoderms.     

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.     

Sublethal predation on Stylaria lacustris: a study of regenerative capabilities

Research on predator–prey interaction has generally ignored the possibility of prey injury by predator. Although injured prey usually constitute a minor group in a population, sublethal predation can play an important role in some aquatic assemblages. In a laboratory experiment, I tested the effect of attack by larvae of the damselfly Ischnura elegans and tanyponid Clinotanypus nervosus on the oligochaete Stylaria lacustris. Predation by these insect larvae caused damage to the prey which then are able to escape and survive. More than 50% of the worms used in the experiment were damaged by C. nervosus. Results of predation by I. elegans larvae of different lengths showed that the number of damaged worms decreased with the length of predatory larvae. Small predators injured more worms than large ones, which killed and totally consumed most of the prey. Damage to S. lacustris usually involves the loss of anterior, posterior or both these fragments (middle part preserved). An analysis of the survival of worms revealed that individuals which lost anterior, posterior, or both fragments survived equally well as control ones, with the exception of worms that lost 70% of the body length posteriorly amputated. It should be noted that these worms were the least numerous in all worms damaged by predators. The laboratory experiment on the regenerative capability of S. lacustris showed that after amputation, all worms regenerated the lost structures and started to increase in length. The small individuals after amputation of both anterior and posterior fragments achieved their initial length in the course of the experiment. It is likely that the regenerative capability in S. lacustris is an adaptation to sublethal effects of predation, which seems to play an important role in littoral assemblages dominated by oligochaetes.     

Serially duplicated regenerates from the anterior half of the axolotl limb after retinoic acid treatment

Summary Axolotl (Ambystoma mexicanum) forearms were divided, by an incision between the radius and ulna, to produce anterior and posterior halves. These were prevented from fusing together again by a graft of head skin and amputated through the wrist. This procedure enabled independent regeneration from both halves of the stump. Anterior half stumps produced a single digit while the posterior halves mainly regenerated three digits, the two halves together making a single hand. Treatment with retinoic acid, injected intraperitoneally four days after amputation, abolished regeneration from the posterior half stump and produced proximo-distally duplicated regenerates from the anterior half. The duplicated regenerates had in most cases a complete four digit hand and were therefore more than proximalised regenerates from the anterior side of the limb. Replacement of anterior limb skin with head skin had no effect on the response of the regenerating limb to retinoic acid. In species where application of retinoic acid induces anterior-posterior duplications, these are always derived from the anterior side of the limb. The results presented here show that the morphogenic effects of retinoic acid in inducing proximo-distal duplications are also due to its effects on the anterior tissues of the limb.Excellent technical assistance was provided by Carole Ross and Marjory Shiach and useful discussion were had with Paul Martin, David Wilson and Gavin Swanson     

The importance of larval eyes in the polychaete Capitella teleta: effects of larval eye deletion on formation of the adult eye

In many marine invertebrates with biphasic life cycles, juvenile/adult traits begin to develop before metamorphosis. For structures that are present at multiple developmental stages, but have distinct larval and adult forms, it is unclear whether larval and adult structures have shared or distinct developmental origins. In this study, we examine the relationship between the larval and adult eyes in the polychaete Capitella teleta. In addition, we describe a novel marker for larval and juvenile photoreceptor cells. Infrared laser deletion of individual micromeres in early embryos suggests that the same micromeres at the eight‐cell stage that are specified to generate the larval eyes also form the adult eyes. Direct deletion of the larval eye, including the pigment cell and the corresponding photoreceptor cell, resulted in a lack of shading pigment cells in juveniles and adults, demonstrating that this structure does not regenerate. However, a sensory photoreceptor cell was present in juveniles following direct larval eye deletions, indicating that larval and adult photoreceptors are separate cells. We propose that the formation of the adult eye in juveniles of C. teleta requires the presence of the pigment cell of the larval eye, but the adult photoreceptor is either recruited from adjacent neural tissue or arises de novo after metamorphosis. These results are different from the development and spatial orientation of larval and adult eyes found in other polychaetes, in which two scenarios have been proposed: larval eyes persist and function as adult eyes; or, distinct pigmented adult eyes begin developing separately from larval eyes prior to metamorphosis.     

Proliferative response of the stem cell system during regeneration of the rostrum in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Platyhelminthes)

Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a.     

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.     

What role do annelid neoblasts play? A comparison of the regeneration patterns in a neoblast-bearing and a neoblast-lacking enchytraeid oligochaete

The term 'neoblast' was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.     

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.     

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.     

The regeneration of axolotl limbs covered by frog skin

Forearm skin of Stage XXIV Rana pipiens, which cannot regenerate limbs, was removed and placed upon the skinned forearms of young axolotls. The axolotl limbs were amputated immediately through the level of the grafts. Frog epidermis migrated to cover the amputation surface. Dedifferentiation and early blastema formation occurred beneath the frog wound epidermis. Limb regeneration continued, but in time axolotl epidermis overgrew the frog epidermis. The experiment shows that epidermis from nonregenerating frog limbs is still capable of supporting typical epimorphic regeneration.     

Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin

Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.     

Regenerative ability of double-half and half upper arms in the newt, Notophthalmus viridescens

The upper arms of adult newts (Notophthalmus viridescens) were surgically manipulated to create double-half dorsal, double-half ventral, double-half anterior, and double-half posterior upper arms, and longitudinal half-dorsal, half-ventral, half-anterior, and half-posterior upper arms. Amputation through the double-half upper arms usually failed to elicit normal distal regeneration, despite the fact that an apparently normal regeneration blastema was initially formed. Instead, regeneration in these cases was limited to the formation of a variable number of small cartilage elements. On the basis of these results it is concluded that a complete limb circumference is required for distal transformation in newts, in addition to the well-established requirements for a wound epidermis, adequate innervation and dedifferentiation leading to blastema formation. A model for the sequential generation of new parts of the limb pattern during distal transformation from a complete circumference is presented. This model can also account for the occurrence of normal early stages of regeneration in double-half upper arms. Half upper arms which were amputated immediately were shown to develop single, complete regenerates. If amputation of half upper arms was delayed three or more weeks to permit complete wound healing, a supernumerary limb from the lateral wound surface sometimes developed in addition to a complete, single limb from the distal amputation surface.