The role of grafted skin in the regeneration of X-irradiated axolotl limbs

The primary aim of these experiments was to follow the cells descended from limb skin through the process of limb regeneration to determine what range of differentiations these cells may assume. Triploid hindlimb or forelimb skin was grafted to the denuded thighs of diploid host axolotls that had previously received 3000 R of X irradiation across both hindlimbs and the intervening pelvic area. The host limbs were then amputated through their grafts and permitted to regenerate. Cartilage, perichondrium, joint connective tissue, general connective tissue, dermis, and epidermis were present in all the regenerated limbs, but only 10% of the regenerates contained muscle. Tabulation of nucleolar numbers showed that the majority of cells in each regenerated tissue originated from the grafted skin. A strong correlation was demonstrated between the forelimb or hindlimb origin of the skin grafts and the number of digits regenerated.     

Cellular behavior in the anteroposterior axis of the regenerating forelimb of the axolotl, Ambystoma mexicanum

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

Intercalation and the cellular origin of supernumerary limbs in Xenopus

The hypothesis that a specialized polarizing zone controls the pattern of the anterior-posterior axis during limb development in Xenopus has been tested by analysing the cellular contribution to supernumerary limbs. Supernumerary limbs were generated by grafting hindlimb buds contralaterally between X. borealis and X. laevis to appose anterior and posterior limb tissues. Cells derived from these two species of Xenopus are readily identified by staining with quinacrine. The analysis of cellular contribution showed that supernumerary limbs consist of approximately half anterior-derived (57%) and half posterior-derived (43%) cells. These data are not consistent with the polarizing zone theory but are consistent with the hypothesis that both supernumerary limbs and normally developing limbs arise from intercalary interactions between limb bud cells with different positional values.     

Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum)

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.     

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.     

Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface.

The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.     

The effect of healing time on the proximodistal organization of double-half forelimb regenerates in the axolotl,Ambystoma mexicanum

The effect of healing on the proximodistal organization of regenerates from double-half forelimbs was studied. Double-anterior and double-posterior upper forelimbs were prepared surgically and amputated at 5, 10, 15, 20, 30, and 60 days after grafting. All experimental groups regenerated hypomorphic skeletal patterns. Double-half forelimbs amputated at Days 5 and 10 regenerated more distally complete skeletal patterns than did limbs amputated at Days 30 and 60. The mean numbers of skeletal elements regenerated were seen to decrease as a function of time after grafting, with the maximal suppression of skeletal patterns observed to occur when limbs were amputated 30 days following grafting. There was no appreciable difference between limbs amputated at Days 30 and 60. These results suggest that healing time has a profound effect on the proximodistal organization of limbs regenerated from double-half forelimb stumps.     

Relationship between innervation and forelimb regenerative capacity in the postmetamorphic pond frog Rana brevipoda porosa.

The limb regenerative capacity and the quantity of innervation (the percentage of a cross-sectional area of amputation forelimb stump occupied by nerves) in the pond frog, Rana brevipoda porosa, was investigated in postmetamorphic froglets and adults of various sizes by means of amputating forelimbs through the zeugopodium. Nearly all the amputated limbs of newly metamorphosed froglets, 18-19 mm in snout-vent length, showed heteromorphic regeneration. However, the larger the body size, the lower the presence of limb regeneration. Limb regenerative capacity was completely lost in froglets and adults with snout-vents larger than 35 mm. The quantity of innervation of limbs was highest in newly metamorphosed froglets, gradually decreasing with growth. The nerve quantity in adults with a snout-vent length between 60-67 mm was approximately half that of the froglets. When the nerve supply was augmented by deviating ipsilateral sciatic nerve bundles to the forelimb stump, almost all limbs, which were usually non-regenerative with normal innervation, regenerated heteromorphically. These results show that the decline in limb regenerative capacity during postmetamorphic growth is in part attributable to the reduction in innervation levels to below the threshold level required for regeneration.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

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.     

Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs

The effects of retinoic acid (RA) on anteroposterior (AP) positional memory of regenerating axolotl limbs were tested after removing the anterior or posterior half from the zeugopodium (lower arm or leg). RA (150 micrograms/g body wt) was injected into groups of animals bearing the following types of limbs: (1) anterior and posterior half zeugopodia grafted to the eyesocket and amputated distally 7 days later; (2) unamputated anterior and posterior half zeugopodia in situ; (3) double anterior and double posterior half zeugopodia amputated distally 7 days after their construction; (4) sham-operated zeugopodia amputated distally 7 days after operation. Controls consisted of these four groups injected with the retinoid solvent, dimethyl sulfoxide, or not injected. Control half zeugopodia grafted to the eyesocket regenerated no more than one or two digits. Control unamputated half zeugopodia in situ underwent partial or complete regeneration of the missing half from the proximal and midline wound surfaces exposed during construction of the half zeugopodia. Control double anterior and posterior zeugopodia both regenerated symmetrical, hypomorphic regenerates with 1-3 digits in the double anteriors and 1-6 digits in the double posteriors. Sham-operated controls regenerated normally. Regenerating anterior and posterior halves responded differently to RA. RA-treated anterior half zeugopodia in the eyesocket, and anterior half stumps adjacent to the unamputated posterior half zeugopodia in situ both produced regenerates that duplicated stump structures in the proximodistal axis and formed a complete and normal AP pattern. RA-treated double anterior zeugopodia regenerated proximodistal-duplicated pairs of mirror-imaged limbs, each with a complete and normal AP pattern. In contrast, half posterior zeugopodia in the eyesocket, the posterior half stumps of unamputated half anterior zeugopodia in situ, and double posterior zeugopodia all failed to regenerate. These results suggest that RA modifies positional memory in only one direction in the AP axis, posterior.     

The role of gap junctions in patterning of the chick limb bud

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.     

Limb Development and Regeneration

Experiments on developing and regenerating vertebrate limbshave led to the idea that pattern formation and growth controlare causally linked. The mechanism by which position-specificgrowth occurs is termed intercalation, and evidence is presentedthat implicates intercalation in the initiation, maintenanceand cessation of growth during limb formation. We conclude thatamong the variety of cell types present in limbs, only fibroblastshave been shown to possess the positional information necessaryfor intercalation. Hence we propose that the limb pattern isgenerated by intercalation between fibroblasts to give riseto a connective tissue scaffold, which in turn dictates thepositioning and morphogenesis of all of the differentiated celltypes of the limb. Finally, we review evidence that regenerativefailure among higher vertebrates is linked to defects in theintrinsic cellular mechanisms of growth control (intercalation)and conclude that progress towards the goal of stimulating regenerativelimb outgrowth in non-regenerating vertebrates will be contingentupon a better understanding of these intrinsic mechanisms.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Percutaneous osseointegrated prostheses for amputees: Limb compensation in a 12-month ovine model

Percutaneous osseointegrated prostheses are being investigated as an alternative strategy to attach prosthetic limbs to patients. Although the use of these implants has shown to be promising in clinical trials, the ability to maintain a skin seal around an osseointegrated implant interface is a major challenge to prevent superficial and deep periprosthetic infections. The specific aim of this study was to establish a translational load-bearing ovine model to assess postoperative limb compensation and gait symmetry following a percutaneous osseointegrated implant. We tested the following hypotheses: (1) the animals would return to pre-amputation limb loads within 12-months; (2) the animals would return to a symmetrical gait pattern (stride length and time in stance) within 12-months. The results demonstrated that one month following surgery, the sheep loaded their amputated limb to a mean value of nearly 80% of their pre-amputation loading condition; by 12-months, this mean had dropped to approximately 74%. There was no statistical differences between the symmetry of the amputated forelimb and the contralateral forelimb at any time point for the animals stride length or the time spent in the stance phase of their gait cycle. Thus, the data showed that while the animals maintained symmetric gait patterns, they did not return to full weight-bearing after 12-months. The results of this study showed that a large animal load-bearing model had a symmetric gait and was weight bearing for up to 12 months. While the current investigation utilizes an ovine model, the data show that osseointegrated implant technology with postoperative follow-up can help our human patients return to symmetric gait and maintain an active lifestyle, leading to an improvement in their quality of life following amputation.     

Nerve-independence of limb regeneration in larval Xenopus laevis is related to the presence of mitogenic factors in early limb tissues.

Early limbs of larval Xenopus laevis can form a regeneration blastema in the absence of nerves. The nerve-independence could be due to the synthesis of neurotrophic-like factors by the limb bud cells. To test this hypothesis, two series of experiments were performed. Series A: the right hindlimbs of stage 57 larvae (acc. to Nieuwkoop and Faber. 1956. Normal table of Xenopus laevis [Daudin]. Amsterdam: North-Holland Pub. Co.), which are nerve-dependent for regeneration, were amputated through the tarsalia. The regenerating limbs were submitted to: sham denervation; denervation; denervation and implantation of a fragment of an early limb, or a late limb, or a spinal cord. Series B: froglets were subjected to amputation of both forelimbs. The cone blastemas were transplanted into denervated hindlimbs of stage 57 larvae, together with a fragment of an early or a late limb. The results in series A showed that the implantation of early limb tissue into the denervated blastema maintained cell proliferation at levels similar to those observed after the implantation of a spinal cord fragment or in sham denervated blastemas. However, the implantation of late limb tissues were ineffective. The results of series B showed that the implantation of early limb tissue, but not of late limb tissue prevented the inhibition of cell proliferation and the regression of denervated limb blastemas of juveniles. These results indicate that the nerve-independence is related to the synthesis of diffusible mitogenic neurotrophic-like factors in early limb tissues, and that nerve-dependence is established when differentiated cells of late limb tissues stop producing these factors.     

Transforming growth factor: beta signaling is essential for limb regeneration in axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-beta). In the present study, the full length sequence of the axolotl TGF-beta1 cDNA was isolated. The spatio-temporal expression pattern of TGF-beta1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-beta signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-beta type I receptor, SB-431542, we show that TGF-beta signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-beta signaling are down-regulated. These data directly implicate TGF-beta signaling in the initiation and control of the regeneration process in axolotls.     

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.     

Positional information in the forelimb of the axolotl: experiments with double-half tissues

It has been demonstrated recently that upper forelimbs of axolotls comprised of symmetrically arranged soft tissues do not regenerate (P. W. Tank, 1978,J. Exp. Zool.204, 325–336). These double-half forelimb stumps contained skin, muscle, and loose connective tissues in symmetrical arrangement. The present study explores the roles of muscle, skin, and epidermis in the regeneration of double-half forelimbs by grafting them separately to create forelimb stumps bearing symmetrical arrangements of these individual tissues. Forelimb stumps bearing symmetrically arranged flexor and extensor muscles and normally arranged skin underwent complete regeneration (96%). Forelimbs comprised of double-half skin overlying normally arranged muscles and deep tissues formed hypomorphic structures and nonregenerates (56%) with some single and multiple regenerates. Limbs with double-half deep tissues and complete epidermis either regenerated distally incomplete patterns (47%), single patterns (33%), or multiple patterns (20%). Those forelimbs comprised of double-half skin and no muscle regenerated incomplete patterns in the majority of cases (56%) but single and multiple limbs also were formed. Based on these results it can be concluded that no single type of tissue is solely responsible for the regenerative failure experienced by double-half forelimbs in the earlier study. The complete failure of forelimb regeneration occurs only when all types of soft tissues tested (skin, muscle, and deep connective tissues) are present in symmetrical arrangement.