The failure of double-half forelimbs to undergo distal transformation following amputation in the axolotl, Ambystoma mexicanum

Although capable of initiating early regenerative responses, axolotl forelimb stumps which are composed of double-half limb tissues fail to undergo the events that normally lead to the replacement of missing parts. In the present study, the posterior halves of right forelimbs were exchanged with the anterior halves of left forelimbs, or the dorsal halves of right forelimbs were exchanged with the ventral halves of left forelimbs. Forelimbs were amputated through the graft region 30 days after grafting. Limb stumps bearing double-dorsal, double-ventral or double-posterior tissues either produced hypomorphic regenerates or failed to form any externally visible outgrowth. When the limb stump bore double-anterior tissues, no externally visible structures were formed. Normal and multiple regenerates were never formed by double-half limbs. These results are discussed in terms of the polar coordinate model and suggest that the regeneration blastema requires a complete circumference of positional values in order to complete distal transformation.     

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.     

The interaction between the blastema and stump in the establishment of the anterior-posterior and proximal-distal organization of the limb regenerate

Interactions between the limb stump and the developing regenerate were studied in the limbs of adult newts, Notophthalmus viridescens. Forelimb blastemas at various stages were transplanted to the contralateral forelimb such that the anterior-posterior axes of stump and blastema were opposed. The blastemas were transplanted either from a proximal to distal, distal to proximal, proximal to proximal, or distal to distal level limb stump. The results indicate that at the earliest stage studied the anterior-posterior axis of the blastema is established but is not stable. An interection between the stump and blastema at this early stage results in the production of a variety of limbs intermediate in polarity between the graft and the stump. At all later stages, the original anterior-posterior axis of the blastema can be retained, although under certain grafting conditions the stump can still exert considerable influence over the anterior-posterior organization of the final regenerate. In those circumstances in which the blastema retains its original handedness, the interaction between stump and blastema results in the production of separate anterior and posterior supernumerary regenerates.The results of transplanting proximal blastemas to a distal limb level indicate that the proximal boundary of the blastema has been established by the earliest stage studied, leading to the production of limbs with serially duplicated segments. However, irrespective of the stage of a blastema transplanted from a distal to proximal level, there are no deleted structures in the proximal-distal axis of the resulting limb. From both histological examination of transplanted regenerates and the arrangement of skeletal elements of the resulting limbs, it is postulated that the stump plays an important role in the production of the intercalary regenerate.     

Interactions between irradiated and unirradiated tissues during supernumerary limb formation in the newt.

The interactions between irradiated and unirradiated blastemas and stumps in the newt forelimb were studied. Irradiated right blastemas at the stage of early digits were grafted to unirradiated left stumps and unirradiated left blastemas were grafted to irradiated right stumps. Grafts were oriented with their anterior-posterior axes opposed to that of the stumps. Supernumerary limbs ranging in completeness from one to four digits were found to arise predominantly on the anterior or posterior sides of the host limb. The graft developed well when the blastema was unirradiated and had reversed handedness with respect to the stump. Irradiated grafts developed poorly. On occasions, limbs with two supernumerary structures were found. The results are discussed in terms of the origin of the cells which comprise the supernumerary limbs and their bearing on a recently presented model concerned with pattern specification and regulation in epimorphic fields.     

Regulation after proximal or distal transposition of limb regeneration blastemas and determination of the proximal boundary of the regenerate.

When blastemas of several stages of differentiation were grafted in normal orientation to stump levels proximal or distal to their level of origin, normal limbs regenerated. Histological and autoradiographic studies of the development of these regulated limbs showed that the grafted blastemas formed only structures normally distal to their level of origin. In the case of a blastema transplanted proximally, regulation occurred by intercalary regeneration from the stump, whereas, when blastemas were transplanted distally, regulation appeared to take place within the blastema itself by a distal shift in its pattern of organization. The results suggest that the proximal limit of the limb regenerate is determined by level-specific properties of the limb cells but that these properties allow for interactions leading to regulation when different levels of stump and blastema are brought together.     

Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.     

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.     

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.     

Blastema cell proliferation in vitro: effects of limb amputation on the mitogenic activity of spinal cord extracts

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.     

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.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Epimorphic vs. tissue regeneration in Xenopus forelimbs.

Postmetamorphic froglets of Xenopus laevis regenerate hypomorphic unbranched spikes from amputated arm stumps. These are composed primarily of cartilage, produced from blastemalike structures sparsely populated with cells and rich in connective tissue. Some consider these outgrowths to be an example of epimorphic regeneration produced from blastemas, albeit deficient ones. Others interpret them as a case of tissue regeneration derived from fibroblastemas augmented by chondrocytes and periosteal and perichondrial fibroblasts. To resolve these alternatives, forelimbs were amputated proximal to the wrist, skinned, and inserted through the body wall into the abdominal cavity. In the absence of skin, epidermal wound healing failed to occur and blastemas could not develop. After 2 months, by which time controls had regenerated spikes averaging 3.38 mm long, the denuded stumps had not given rise to outgrowths. They typically developed cartilaginous caps on the severed ends of the radius-ulna, and in rare cases formed amorphous growths of cartilage. If blastema formation is considered diagnostic of epimorphic regeneration and tissue regeneration can proceed in the absence of epidermal wound healing and blastema formation, these findings lead to the conclusion that Xenopus limb regeneration is epimorphic.     

Regenerative responses in cultured hindlimb stumps of larval Xenopus laevis.

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8) mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of positional identity of axolotl blastema cells in vitro

Summary Previous grafting experiments have demonstrated that cells from non-contiguous positions within developing and regenerating limbs differ in a property referred to as positional identity. The goal of this study was to determine how long the positional identity of axolotl limb blastema cells is stable during culture in vitro. We have developed an assay for posterior positional properties such that blastema cells can be cultured and then grafted into anterior positions in host blastemas, to determine if they can stimulate supernumerary digit formation. We report that posterior blastema cells are able to maintain their positional identities for at least a week in culture. In addition, we observed that blastema cells are able to rapidly degrade collagenous substrates in vitro, a property that apparently distinguishes them from limb cells of other vertebrates. These results provide information regarding the time boundaries within which the positional properties of blastema cells can be studied and manipulated in vitro. Correspondence to: S.V. Bryant     

Supernumerary limgs in amphibians: experimental production in Notophthalmus viridescens and a new interpretation of their formation.

The formation of supernumerary limbs was studied in the adult newt, Notophthalmus viridescens. Forelimb blastemas at the stages of medium bud and early digits were either transplanted to the contralateral forelimb with their dorsal-ventral axis opposed to that of the limb stump, or removed, rotated through 180°, and replaced on the same limb stump with both dorsal-ventral and anterior-posterior axes opposed to those of the stump, or as a control, removed, and replaced in normal orientation. Supernumerary limbs were produced in both experimental series, but not in the controls.Following contralateral transplantation, supernumerary limbs arose close to the graft junction at the two positions where dorsal limb tissue was in contact with ventral limb tissue. Both dorsal and ventral supernumerary limbs were of the same handedness as the limb stump and they were mirror-images of the regenerate developing directly from the transplanted blastema. Following 180° rotation, supernumerary limbs arose close to the graft junction at those positions where anterior-ventral and posterior-dorsal limb tissues were in contact. The supernumerary limb which arose in the posterior-dorsal position with respect to the limb stump was a mirror-image of the transplant, and was therefore of opposite handedness to both transplant and stump. The supernumerary limb which arose in the anterior-ventral position was of the same handedness as both transplant and stump. A new model of pattern regulation in epimorphic fields which can account for these results and which has retrospective value in the interpretation of earlier experiments on developing limbs is discussed.     

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

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

The irradiated epidermis inhibits newt limb regeneration by preventing blastema growth. A histological study

It has been established that X-ray irradiation localized to a forelimb or entire irradiation of premetamorphic Pleurodeles larvae prevented limb regeneration. Transplantation of non-irradiated skin, dermis or muscle to limb stumps of locally irradiated newts was sufficient to allow a blastema to develop. Transplantation of the same tissues to limb stumps of entirely irradiated newts yielded different results with the different graft types. Skin graft allowed a normal blastema to be established but dermis or muscle grafts did not. In order to define more precisely the role played by the epidermis in the establishment of a blastema, and in the growth of a regenerate, different combinations of limb tissues, either irradiated or not, were carried out at the level of amputated limb stumps. At four different times (8-10 days; 13-15 days; 20-23 days; 30 days or more) after amputation the stumps were examined in histological longitudinal sections to study the first events of regeneration, that is dedifferentiation and growth. Dedifferentiation occurred in both normal and irradiated tissues of mesodermal origin. The healthy mesenchymal cells began dividing and formed a growing blastema only when associated with a non-irradiated epidermis. Healthy mesenchymal cells covered with an irradiated epidermis exhibited a few mitoses after dedifferentiation, but the mitotic figures became rarer and rarer until the animals died. The lack of dense accumulation of blastemal cells in such limb stumps suggested that the healthy epidermis allows the mesenchymal cells to divide actively to constitute a growing blastema. Hence, X-ray irradiation seems to be responsible for the loss of such an epidermal mitogenic influence on the underlying mesenchymal cells.     

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated,nerve‐dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF‐like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine‐rich domain (CRD‐NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD‐NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve‐dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve‐dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000     

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.