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.     

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.     

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.     

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.     

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.     

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.     

The influence of denervation on grafted hindlimb regeneration of larval Xenopus laevis

The aim of the present research is to ascertain whether in larval Xenopus laevis nerve-independence for the regeneration of early stage limbs and nerve-dependence of late stage limbs observed in a previous work (Filoni and Paglialunga, '90) is related to extrinsic (systemic) factors or to intrinsic changes taking place in the limb cells themselves during development. In this paper the regenerative capacity of early and late stage hindlimbs under the same extrinsic conditions, insofar as both are grafted onto the denervated hindlimbs of host larvae at the same developmental stage, is studied. All the grafted limbs are amputated after the host larvae have reached stage 57-58 (according to Nieuwkoop and Faber, '56). In experiment I, the grafted limb is amputated at stage 52, at the thigh level; in experiment II, the grafted limb is amputated at stage 54-55, at the tarsalia level; in experiment III the grafted limb is amputated at stage 57, at the tarsalia level. In all three experiments, together with the grafted limb, also the host limb is amputated at the tarsalia level. The results show that while grafted limbs amputated at stages 52 and 54-55 regenerate in the absence of nerves, grafted limbs amputated at stage 57 cannot. The failure of late stage grafted limbs to regenerate cannot be explained in terms of an immune-type inhibiting reaction since it has been observed also in denervated autografted limbs and in the host limbs. Since all the grafted limbs are in the same environmental conditions, the results show that in larval Xenopus laevis nerve-independence for regeneration of early stage limbs and nerve-dependence of late stage limbs are not related to factors extrinsic to the limb but to intrinsic changes taking place in the limb cells themselves during development.     

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。     

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.     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

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.     

Regeneration of Soft Tissues Is Promoted by MMP1 Treatment after Digit Amputation in Mice

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice.     

Autoradiographic analyses of35S-sulfate uptake in regenerating limbs of larvalAmbystoma

Summary Autoradiographic and histochemical techniques were used to determine whether chondrocytes continue to synthesize chondroitin sulfate or closely related compounds during morphological dedifferentiation of these cells in regenerating limbs of larvalAmbystoma. Forelimbs were amputated either through the mid-diaphysis or the distal epiphysis of the humerus and each animal was subsequently injected with Na₂ ³⁵SO₄ at an appropriate stage of regeneration. Incorporation of the isotope and metachromatic staining responses were used as indices of cell specialization.In autoradiographs of unamputated limbs, epiphyseal chondrocytes exhibited moderate sulfate incorporation, whereas isotope uptake was slight in diaphyseal regions. Accordingly, in early stages of regeneration, limbs amputated through the diaphysis showed a low level of sulfate incorporation by cartilage-derived cells; since these cells dispersed during blastema formation, they were not identifiable in later stages. When limbs were amputated through the epiphysis, the matrix here underwent slow dissolution and epiphyseal-derived chondrocytes and their progeny consequently remained identifiable as they contributed to the blastema. These cells continued to exhibit isotope uptake, even during early and middle stages of regeneration —results which support the idea of tissue-specific regeneration of cartilage.Further inspection of the stained autoradiographs revealed that in addition to chondrocytes and blastema cells derived from chondrocytes, fibroblast-like cells located lateral to the limb skeleton and seemingly derived from muscle or muscle-associated cells also exhibited a moderate label during certain stages in the restoration of the limb. In several respects isotope incorporation and related metachromatic responses by these two types of cells during blastemal and early redifferentiating stages of regeneration were seen to parallel results reported in the literature of histochemical and autoradiographic studies of differentiating chick fimb buds. These observations, which may be added to previous analogies concerning developing and regenerating limbs, suggest a similar mechanism of cytodifferentiation in the two systems. The possibility is also considered that the observed isotope uptake by cells of non-cartilaginous origin may indicate the synthesis of sulfated glycosaminoglycans which alfect cell interactions during the regenerative processes.A portion of a dissertation submitted to the University of New Hampshire in partial fulfillment of the requirements for the degree of Doctor of Philosophy.NASA Predoctoral Trainee during the course of this work.The authors wish to thank Nr. Carl Paulitz for his assistance in photography of the autoradiographs.     

Fgfs control homeostatic regeneration in adult zebrafish fins

Adult teleost fish and urodele amphibians possess a spectacular ability to regenerate amputated appendages, based on formation and maintenance of progenitor tissue called a blastema. Although injury-induced, or facultative, appendage regeneration has been studied extensively, the extent to which homeostatic regeneration maintains these structures has not been examined. Here, we found that transgenic inhibition of Fgf receptors in uninjured zebrafish caused severe atrophy of all fin types within 2 months, revealing a requirement for Fgfs to preserve dermal bone, joint structures and supporting tissues. Appendage maintenance involved low-level expression of markers of blastema-based regeneration, focused in distal structures displaying recurrent cell death and proliferation. Conditional mutations in the ligand Fgf20a and the kinase Mps1, factors crucial for regeneration of amputated fins, also caused rapid, progressive loss of fin structures in otherwise uninjured animals. Our experiments reveal that the facultative machinery that regenerates amputated teleost fins also has a surprisingly vigorous role in homeostatic regeneration.     

The extent of regeneration is impacted by the stage of amputation in ladybird beetles: A case study in Cheilomenes sexmaculata

Regeneration has been a topic of interest across a range of taxa for centuries, and arthropods are no exception. Trade-offs associated with regeneration are likely to involve the reallocation of resources away from other metabolic activities such as growth, development or reproduction. This might be reflected in costs to some developmental traits of the organism, despite regeneration being advantageous. These associated costs might also differ with the stage of injury or amputation. Here, we hypothesise that the extent of regeneration and trade-offs associated with it may be stage-specific. To test this hypothesis, the right forelimb of four larval stages of the ladybird beetle Cheilomenes sexmaculata (Fabricius) (Coleoptera: Coccinellidae) was amputated. Amputated individuals were reared until adulthood, and all developmental transitions were recorded. Regenerated legs in all the treatments were smaller than the controls, which did not experience the amputation, and the regenerative potency of early larval stages was higher than that of late larval stages. Limb regeneration caused delays in post-amputation developmental duration in all the treatments, increasing their total developmental period. The length of the unamputated left foreleg as well as the wing and antenna size were also reduced in regenerated beetles, showing some internal trade-off. However, there were no significant differences observed between regenerated and control adults in their fresh body weight and body size. Thus, limb regeneration depends upon the stage of larval development at which the amputation was performed. Amputation also affects the development of other appendages. The delay in normal beetle development might have been observed because of extra resource requirement, their allocation as well as reprogramming of the expression of some genes during regeneration.     

Cellular contribution to symmetrical forelimbs from triploid-marked "polarizing region" in the embryo of axolotl, Ambystoma mexicanum

Grafts of posterior tissue placed anterior to the limb bud in the salamander embryo exert a polarizing influence. To explain this result, the idea that the anteroposterior axis of the developing forelimb is polarized by a diffusible morphogen has been proposed. An alternative hypothesis, and the working hypothesis of the present study, is that the polarization of the developing salamander forelimb is accomplished by short-range cellular interactions resulting in intercalation rather than by the more global influence of a diffusible morphogen. One prediction of this intercalation hypothesis is that cells will be contributed to the limb from the "polarizing tissue." To test this idea, grafts of triploid marked polarizing tissue were implanted anterior to the limb bud in 82 diploid axolotl embryos at stages 32-34 of development. A total of 27 (33%) of the limbs that resulted were symmetrical and ranged in complexity from one to seven digits. Histological analysis of a subgroup of the original symmetrical limbs revealed that mesodermally derived tissues in the anterior side of these limbs (the side which formed as a duplication in response to the influence of the graft) contained high percentages of trinucleolate cells (muscle, 12.1%; connective tissue tissue, 12.5%; and cartilage, 13.4%) when compared to similar tissues in the posterior side of the same symmetrical limbs (muscle, 1.8%; connective tissue , 0.7%; and cartilage, 0.6%). When symmetrical limbs were amputated, 73% regenerated symmetrical limbs. When these regenerated limbs were again amputated, 63% formed symmetrical secondary regenerates. Histological analysis of the first generation of regenerated limbs revealed that the pattern of distribution of trinucleolate cells in each regenerate was similar to the pattern seen in the original symmetrical limb. These results indicate that there is considerable cellular contribution to the anterior side of the symmetrical forelimb from the mesoderm of grafted "polarizing tissue." This result supports the idea that short-range cellular interaction are sufficient for formation of symmetrical forelimbs in salamander embryos.     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.