Tail regeneration and ependymal outgrowth in the adult newt, Notophthalmus viridescens, are adversely affected by experimentally produced ischemia

Spinal axons of the adult newt will regenerate when the spinal cord is severed or when the tail is amputated. Ischemia and associated hypoxia have been correlated with poor central nervous system regeneration in mammals. To test the effects of ischemia on newt spinal cord regeneration, the spinal cord and major blood vessels of the newt tail were severed 2 cm caudal to the cloaca as a primary injury. This primary injury severely reduced circulation in the caudal direction for 7 days; by day 8, circulation was largely restored. After various periods of time after primary injury, tails were amputated 1 cm caudal to the primary injury (in the area of ischemia) and tested for regeneration. If the tail was amputated within 5 days of the primary injury, regeneration did not occur. If amputation was 7 days or longer after the primary injury, a regenerative response occurred. Histology showed that in the non-regenerating tails the spinal cord and associated ependyma, known to be important to tail regeneration, had degenerated in the rostral direction. Such degeneration was prevented when tails were first amputated and allowed to form blastemas before the primary injury. The data indicate that the first 5-7 days of blastema formation are particularly sensitive to compromised blood flow (ischemia/hypoxia). It follows that mechanisms must be present in the adult newt to reduce ischemia to a minimum and thus allow ependymal outgrowth and tail regeneration.     

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.     

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.     

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.     

Morphogenetic control during tail regeneration in Plethodon cinereus: the role of skeletal muscle

The caudal myofibers of Plethodon cinereus do not appear to participate directly in epimorphic tail regeneration following either autotomy or surgical amputation of the tail. The possibility that tail musculature might indirectly influence morphogenesis of the regenerate was tested by unilaterally removing 99% of the lateral muscle mass for five to six caudal segments. Ten days after muscle ablation, tails were amputated through the deficient area. Unlike previous experiences with ambystomid larvae, P. cinereus regulates completely producing a normal tail regenerate and at a rate comparable to that following simple amputation.     

Effects of an augmented nerve supply on forelimb regeneration in the adult mud frog, Rana rugosa.

Forelimbs of the adult mud frog Rana rugosa, when amputated midway through the zeugopodium, regenerate heteromorphically. The resulting regenerative outgrowths were mostly rod shaped and consisted of a cartilaginous core, in which the base was ossified, and muscle elongated distally along the cartilage, the whole being covered by connective tissue and skin. The tip of the regenerating muscle reached a point distally about one third of the length of the regenerative outgrowths. When the innervation of forelimb stumps was augmented by surgical diversion of the ipsilateral sciatic nerve, the amputated limbs regenerated mostly as spatula-shaped outgrowths, which were longer than those of normally innervated forelimbs. Such hyperinnervated regenerates exhibited less ossification of cartilage, or sometimes none at all. However, the regeneration of muscle was more extensive. That is, it reached more than half way along the regenerative outgrowth. Furthermore, denervation resulted in the absence of regeneration in all cases examined. These results clearly indicate that limb regeneration in Rana rugosa is dependent upon the degree of innervation, not only for the early stages of regeneration, but also for the growth and differentiation of the regenerative outgrowth.     

The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations

Background

The zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration.

Methodology/Principal Findings

We show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/ß-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/ß-catenin signalling inhibition and re-amputation.

Conclusions/Significance

We show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/ß-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/ß-catenin signalling for their survival.     

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.     

Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

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.     

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.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Regenerative capability in the hindlimb of Xenopus laevis during ontogenetic development

The regenerative capacity of hindlimb of Xenopus laevis was investigated by amputating the limbs at four levels in various developmental stages including younger postmetamorphosed froglets. Amputations of limbs were performed at the base of limb in stages 50, 51, 52, 53, 54, 55, 58, and 60 (Nieuwkoop and Faber's table), at the middle of limb bud in stages 50, 51, 52 and 54, and at mid-thigh and mid-shank in stages 58 and 60, and the froglets in 2 and 3 cm in snout-vent length. In the present experiments the regenerative capacity of limbs was expressed by the rate of regeneration and morphogenesis. Tadpoles in the stages after 55 failed to regenerate when the limbs were amputated at base level, but individuals in all the other experimental series exhibited regeneration in various rates irrespective of the level of amputation and the stage. The regenerative capacity increased distally along the proximo-distal axis of the limb when amputated at the same stage, while regeneration was better in younger stages than that in older stages when amputations were made at the same levels. The regenerates obtained by amputation of limbs in stages between 50 and 54, were mainly digitated in that they had 5 toes with 3 claws which is the same pattern with the normal limb, 4 toes with 2 claws, 3 toes with 2 claws or one, and 2 toes with one claw etc. Tadpoles at stage 50 could regenerate toes and claws without defect, but in the later the regenerative capacity gradually declined by reducing the number of toes and claws and accompanied by malformation of skeleton as the stage proceeded. The tadpoles in stages after 58, and the froglets of 2 and 3 cm, produced various types of heteromorphic regenerates of shapes such as cone, spike or rod of which the centra were occupied with cartilage rods. However these regenerates showed no morphological differences according to the developmental stages. These heteromorphic regenerates continued their growth even after one year without any sign of development of digitated feet.     

Cell lineage tracing during Xenopus tail regeneration

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.     

Effects of Tail Clipping on Larval Performance and Tail Regeneration Rates in the Near Eastern Fire Salamander,Salamandra infraimmaculata

Tail-tip clipping is a common technique for collecting tissue samples from amphibian larvae and adults. Surprisingly, studies of this invasive sampling procedure or of natural tail clipping – i.e., bites inflicted by predators including conspecifics - on the performance and fitness of aquatic larval stages of urodeles are scarce. We conducted two studies in which we assessed the effects of posterior tail clipping (~30 percent of tail) on Near Eastern fire salamander (Salamandra infraimmaculata) larvae. In a laboratory study, we checked regeneration rates of posterior tail-tip clipping at different ages. Regeneration rates were hump-shaped, peaking at the age of ~30 days and then decreasing. This variation in tail regeneration rates suggests tradeoffs in resource allocation between regeneration and somatic growth during early and advanced development. In an outdoor artificial pond experiment, under constant larval densities, we assessed how tail clipping of newborn larvae affects survival to, time to, and size at metamorphosis. Repeated measures ANOVA on mean larval survival per pond revealed no effect of tail clipping. Tail clipping had correspondingly no effect on larval growth and development expressed in size (mass and snout-vent length) at, and time to, metamorphosis. We conclude that despite the given variation in tail regeneration rates throughout larval ontogeny, clipping of 30% percent of the posterior tail area seems to have no adverse effects on larval fitness and survival. We suggest that future use of this imperative tool for the study of amphibian should take into account larval developmental stage during the time of application and not just the relative size of the clipped tail sample.     

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.     

Analysis of gene expressions during Xenopus forelimb regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

中华真地鳖的断足再生

报道了中华真地鳖Eupolyphaga sinensis Walker的断足再生特征。研究结果表明,不同虫龄期的若虫都有断足再生能力;足的不同部位断足后均能再生;断掉不同数量的足后,只要能成活均可再生。断足再生后,继续断掉再生足的原位或其他部位也可以再生。再生足的跗节均比正常的少一节,具有再生不完整性。断足后,只要经1～2次蜕皮,均可再生。断掉一对足的腿节后,再生足出现大小不一的现象,小的一般发育不全,断足数量多容易出现再生足发育不全。再生足比正常足要小,但生长速度要快,断掉足的腿节或跗节后的再生足经过2次蜕皮后基本可恢复到正常足大小。     

Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)