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.     

Dorsal root ganglia grafts stimulate regeneration of denervated urodele forelimbs: timing of graft implantation with respect to denervation

Amphibian forelimb regeneration is a nerve-dependent process; nerves presumably release one or more neurotrophic factors that stimulate blastema cell division. To date several candidate molecules/factors have been shown to stimulate macromolecular synthesis and/or mitosis but sustained cell cycle activity and blastema development have not been achieved. Because dorsal root ganglia (DRG) implants are capable of promoting regeneration of denervated adult newt limbs (Kamrin & Singer, 1959), we have evaluated the DRG stimulation of regeneration in denervated limbs of adult newts and larval axolotls; two alternative timing strategies were tested as a step toward defining bioassay parameters that best reflect neurotrophic activity. The frequency of regeneration in denervated adult newt limbs was compared after providing DRG before or at the time of denervation (to maintain neurotrophic and cell cycle activity) versus DRG implantation at various postdenervation times (to resupply neurotrophic activity and restimulate suppressed cell cycle activity). The results show that denervated adult newt limbs regenerated most frequently using the maintenance strategy, but as the denervation interval was extended in the restimulation strategy, the frequency of regeneration declined. Larval axolotl limbs responded positively in both maintenance and restimulation DRG-grafting protocols. These results suggest that the efficacy of DRG stimulation of regeneration in adult newts was related to the relative number of blastema cells present at the time of denervation and the proliferative status of the blastema cells; bioassays with denervated adult newt limbs should be designed with these constraints in mind. Because such constraints are not as problematic with the larval axolotl, this species may provide the best opportunity for further defining bioassay parameters related to the neurotrophic stimulation of regeneration.     

Punctuated cell cycling in the regeneration blastema of urodele amphibians

Abstract. We previously assumed that all cells in the regeneration blastema are randomly distributed throughout the cell cycle and actively cycling towards the next mitotic division. We now show that data from continuous labeling (³H-thymidine) experiments do not support this view and favor instead the hypothesis that the blastema cell cycle is punctuated in the g₁ phase wherein cells can enter what we term a transiently quiescent (TQ) position. We call this hypothesis the punctuated cycling (PC) hypothesis. We further propose that the relative sizes of the quiescent and actively cycling populations explain (1) variations in rates of regeneration in different sizes of urodele amphibians, (2) the rate and success of regeneration in different species, and (3) how various controlling factors, such as injury, nerves, growth factors, wound epidermis, and hormones, influence the initiation and progression of the regeneration process. This PC hypothesis is important for interpreting previous pulse labeling data, is consistent with recently obtained continuous labeling data, and is experimentally testable.     

Rescue of blocked cells by reinnervation in denervated forelimb stumps of larval Ambystoma

Cells of amputated, denervated larval Ambystoma forelimbs dedifferentiate and enter the cell cycle but do not subsequently proliferate sufficiently to form a blastema. The denervated limb stump resorbs slowly until reinnervation stimulates regeneration. We used this system to investigate the fate of cells in denervated limbs which undergo early but limited cycling in response to amputation. In Experiment 1, cells were labeled with [3H]thymidine (3H-T) on Day 4 postamputation (PA)/Day 3 postdenervation (PD). Labeled cells were still present on Day 7 PA, but were less frequently observed on Day 13 PA when the limbs were reinnervated and beginning to regenerate. In Experiment 2 we denervated 1 day preamputation to obtain earlier reinnervation and prevent loss of Day 4 PA labeled cells. Cells labeled with 3H-T on Day 4 PA/Day 5 PD were present throughout the denervation period and most were still present on Day 13 PA. Little or no mitotic activity was found among the labeled cells after the initial round of cycling. The apparent cell cycle block was released upon reinnervation on Days 12 and 13 PA when cycling resumed. Labeled mitotic figures were present on Day 13 PA, and the mitotic index of the labeled population increased as a result of reinnervation. These results demonstrate that blocked cells are rescued by nerves, re-enter the cell cycle, and thus contribute to the reinnervation blastema.     

Lack of proliferative response in denervated, reamputated limb regenerates of larval Ambystoma

This paper describes the response of early four-digit regenerates of axolotls to reamputation and denervation. Reamputation of innervated regenerates led to sharp increases in 3H-thymidine labeling index (LI) and mitotic index (MI) on days 2-5 post-reamputation. This resembles the response of innervated limbs following initial amputation. Regenerates that were denervated at the time of reamputation exhibited no proliferative response through day 5. This is in marked contrast to denervated, original amputation limb stumps, in which LI and MI rise for several days (as in innervated stumps) before falling to background levels. Although myelin was scarce near the level of reamputation, the lack of proliferation cannot be explained solely on that basis. The results are consistent with the possibility that the "neurotrophic factor" that causes stump and blastema cell mitosis is not present in unamputated limbs but is made in response to amputation.     

Msx-1 acts as a regulator for blastema growth

Msx-1 is known to regulate ectoderm-mesenchyme and mesenchyme-mesenchyme tissue interactions in the developing vertebrate limb bud. In this study, the spatial and temporal expression pattern ofMsx-1 in the regenerating limbs of axolotls (Ambystoma mexicanum) was examined by whole mountin situ hybridization and RT-PCR. In addition, the effects of retinoic acid (RA) and denervation on theMsx-1 expression were examined as well. In the regenerating normal limbs, the weak expression ofMsx-1 was detected in the mesenchymal cells under the apical ectodermal cap. From the early bud stage, the elevated level ofMsx-1 expression was maintained until the early digit stage. However,Msx-1 expression was rapidly declined by the completion of regeneration. Compared to the normal limb regeneration,Msx-1 expression in RA treated limb regenerates was low until 9 days after RA treatment but increased during blastema growth period both m the lower and the upper arm regenerates. These results suggest that theMsx-1 regulates the proliferation and growth of blastema cells, and its expression may not be responsible for the dedifferentiation process. On the other hand, high level ofMsx-1 expression was noted in the limb stump tissue after denervation. This result indicates thatMsx-1 is also related to the regression of denervated mature tissue.     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.     

Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.     

Cell cycle controls and the role of nerves and the regenerate epithelium in urodele forelimb regeneration: possible modifications of basic concepts

Data from pulse and continuous labeling with [3H]thymidine and from studies with monoclonal antibody WE3 have led to the modification of existing models and established concepts pertinent to understanding limb regeneration. Not all cells of the adult newt blastema are randomly distributed and actively progressing through the cell cycle. Instead, many cells are in a position that we have designated transient quiescence (TQ) and are not actively cycling. We postulate that cells regularly leave the TQ population and enter the actively cycling population and vice versa. The size of the TQ population may be at least partly determined by the quantity of limb innervation. Larval Ambystoma may have only a small or nonexisting TQ, thus accounting for their rapid rate of regeneration. Examination of reactivity of monoclonal antibody WE3 suggests that the early wound epithelium, which is derived from skin epidermis, is later replaced by cells from skin glands concomitant with blastema formation. WE3 provides a useful tool to further investigate the regenerate epithelium.     

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.     

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.     

Stimulation of lens regeneration from the newt dorsal iris when implanted into the blastema of the regenerating limb

Dorsal iris from the eyes of adult Notophthalmus viridescens was transplanted into the blastema of regenerating limbs, subcutaneously in the limb or shoulder region, into the dorsal fin of larval newts and into the hindbrain of larval Ambystoma maculatum. The iris implants into the blastema regenerated lens vesicles or lenses with fibers in 40–75% of the cases. Multiple lenses were found in a few instances. No lenses developed from iris implants into the dorsal fin. Twenty percent of subcutaneous implants of iris formed lenses or lens vesicles, but lens regeneration from implants into the brain occurred only rarely. Denervation of the limb at the time of iris transplantation into the blastema greatly reduced the number of lenses regenerated. Studies on nerve fiber distribution in dorsal fin, subcutaneous areas, and denervated and innervated regenerating limbs, using the Bodian method, showed a general correlation between density of nerve fibers in the implant site and the incidence of lens regeneration from iris implants into that site. These results provide some evidence for a trophic action of nerve fibers on lens regeneration from the iris.     

Lens formation from the cornea following implantation into hindlimbs of larval Xenopus laevis: the influence of limb innervation and extent of differentiation

Corneal fragments of larval Xenopus laevis at stage 48 (according to Nieuwkoop and Faber, '56), were implanted into sham denervated unamputated hindlimbs, denervated unamputated hindlimbs, amputated and sham denervated hindlimbs, and amputated and denervated hindlimbs of larvae at stages 52 and 57. The results show that unamputated limbs at stage 52, either innervated or denervated, manifest a weak capacity to promote the first lens-forming transformations of the outer cornea. This capacity is absent in both limb types at stage 57. After amputation, limbs of both early and late stages form a regenerative blastema and support lens formation from the outer cornea. Denervation of early stage limbs has no appreciable effect on blastema formation and lens-forming transformation of corneal implants. However, denervation of late stage limbs inhibits both processes. These results indicate that the limb tissues of the early stage limbs contain non-neural inductive factors at a low level and that after limb amputation and blastema formation the level of these factors becomes high enough to promote lens formation from implanted cornea, even after denervation. In contrast, the limb tissues of late stage limbs do not contain a suitable level of non-neural inductive factors.     

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.     

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     

Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration

Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the 'aneurogenic' limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).     

Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration

Adult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/β-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.     

Influence of denervation on the regeneration of Pleurodele limbs

Abstract. A cytophotometric study of Feulgen-stained mesenchymal cell nuclei from regeneration blastemas of both innervated and denervated limbs over the 1st 7 days following the midbud stage showed a diminution of the percentage of cells in the S + G₂ phases and a corresponding augmentation of the percentage of cells in the G₀+ G₁ phases. This change, which was temporally correlated with the redifferentiation of the innervated blastemas, was greater in denervated blastemas, even though they do not redifferentiate. From these results, it is concluded that the denervation of midbud blastemas brings about either an extension of the G₁ phase or an exiting from the cell cycle to G₁ (G_0–1), or both phenomena.