Axolotl Limb Research Articles

Regional differences in the distribution of myogenic and chondrogenic cells in axolotl limb blastemas

AbstractThe distribution of myogenic and chondrogenic cells was determined for axolotl limb blastemas at the medium bud stage, before precartilage condensation occurred. Myogenic potential of midstylopod (upper) and distal‐zeugopod (lower) amputation level blastemas, divided into distal, proximal‐core, and proximal‐peripheral regions, was assayed in vitro. Significantly more myonuclei were observed in upper level proximal‐periphery cultures than in ones from any of the other regions. Since cartilage cells give rise almost exclusively to cartilage in the regenerate (Steen, 1968), a heritable cell marker, triploidy, was used to trace the location of chondrogenic cells in histological sections of upper level early and medium bud blastemas. Although there was mixing of the graft cartilage‐derived cells and host soft‐tissue‐derived cells, the previous cartilage cells were found more frequently within the core region at early and medium bud stages. The results of these studies indicate that more chondrogenic cells are present in the core from a very early stage, and that more myogenic cells are present in the proximal periphery by the medium bud stage.

Improved techniques for use of the triploid cell marker in the axolotl, Ambystoma mexicanum.

Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker in the axolotl.

Does vitamin A act on pattern formation via the epidermis or the mesoderm?

AbstractTo determine whether the effect of vitamin A on pattern formation in the regenerating axolotl limb was due to a change in the blastemal mesoderm or the epidermis, tissue grafts were exchanged between normal and retinoic acid‐treated animals. The results were conclusive. The combination of treated epidermis and untreated mesoderm resulted in normal regenerates. The combination of treated mesoderm and untreated epidermis produced regenerates that were serially duplicated in the proximodistal axis just as the controls were. We may, therefore, conclude that vitamin A acts on the mesodermal cells of the blastema in causing proximodistal duplications.

The effect of vitamin A on the regenerating axolotl limb

These experiments describe further investigations into the effects of vitamin A on regenerating limbs. The effects of different retinoids, the time of administration, concentration of vitamin A and histological, autoradiographic and histochemical studies are reported. The most obvious result of vitamin A treatment is to cause proximal elements to regenerate from distal amputation levels, that is to cause serial reduplication of pattern in the proximodistal axis. Retinoic acid was the most potent of the analogues tested and longer times of administration or higher concentrations cause a greater amount of serial reduplication. Various tissue changes have been found which include the inhibition of cell division, loss of cartilage metachromasia, changes in the mucous-secreting properties of the epidermis and an increased packing in the blastemal cells. The significance of these cellular effects in relation to the pattern-formation changes is discussed.

The effect of vitamin A on limb regeneration in Rana temporaria

Previous experiments in which vitamin A has been administered to developing or regenerating limbs have shown that different limb axes are affected. In regenerating axolotl limbs, serial reduplications in the proximodistal axis are produced. In the developing chick limb bud, mirror-imaged reduplications in the anteroposterior axis are produced. Results reported here on Rana temporaria limb buds reveal that vitamin A causes both effects to occur. That is, limbs are both serially reduplicated in the proximodistal axis and mirror imaged in the anteroposterior axis. Time and concentration effects are explored and the significance of these results for our current understanding of axial organisation in limbs is discussed.

Muscle and cartilage differentiation in axolotl limb regeneration blastema cultures.

A tissue culture system is described for explants of mesenchyme from Ambystoma mexicanum limb regeneration blastemas. Explants were cultured on collagen substrate for 3 weeks in minimal essential medium supplemented with the hormones insulin, thyroxine, somatotropin, and hydrocortisone, plus beef embryo extract (EE), 2%. This medium supported extensive cell migration onto the substrate followed by cell proliferation and differentiation of both cartilage matrix and myotubes. Cultures on plastic substrate, rather than on collagen, displayed similar cell outgrowth and cartilage formation, but relatively little myotube formation. Differentiation in EE-supplemented medium was compared with that in two defined media: Explants in medium containing only the hormones showed little outgrowth or cartilage development and never formed myotubes; medium containing the hormones plus fibroblast growth factor, 50 ng/ml, supported an intermediate degree of outgrowth and cartilage development and occasional myotube formation. Explant size was also a factor: Smaller explants survived and formed myotubes less frequently, even when on collagen in EE-supplemented medium.

Increase in mitotic activity of regenerating axolotl limbs by growth factor‐impregnated implants

AbstractThis study was designed to investigate the effects of pituitary fibroblast growth factor (FGF), endothelial cell growth supplement (ECGS), and human serum albumin (HSA) on the mitotic indices of dedifferentiating stump cells in regenerating limbs deprived of a wound epidermis. These cells would normally contribute to blastema cell formation but have been prevented from doing so by the placement of a full thickness skin flap over the amputation site. FGF, ECGS, and HSA were released from copolymer implants inserted between full thickness skin flaps and tibia‐fibula amputation sites in axolotls, Ambystoma mexicanum. The implants were plain or contained either FGF or ECGS or HSA. After 2 wk the mitotic indices of the area of dedifferentiation within these experimental limbs were determined and compared to the mitotic indices of normal regeneration blastemas. Histological analysis revealed that the technique of suturing the full thickness skin flaps into position prevented the formation of dermis‐free areas that can permit, regeneration. Full thickness skin flaps alone, or with a plain implant, or with an HSA‐impregnated implant completely inhibited blastema formation in axolotl hindlimbs. The mitotic indices of these animals showed significant decreases compared to mitotic indices of normal regeneration blastemas. The mitotic indices of animals treated with full thickness skin flaps and implants impregnated with either FGF or ECGS showed significant increases compared to those of normal regeneration blastemas. Precocious cartilage development was found in limbs which contained FGF implants.

Axial organization of the regenerating limb : asymmetrical behaviour following skin transplantation

An extensive series of skin grafting operations has been performed to investigate axial organization in the regenerating axolotl limb. Semicircular cuffs of skin from either anterior, posterior, dorsal or ventral surfaces were exchanged between right and left limbs thereby creating limbs with double anterior, double posterior, double dorsal or double ventral skin, all with normal internal tissues. Both fore and hindlimbs were used at both upper and lower limb levels. Following amputation through the grafted region the resulting regenerates were analysed both by whole-mount cartilage staining to observe the pattern of digits and by serial sectioning to observe the pattern of muscles. There were clear asymmetries in ability to produce duplications - posterior to anterior grafts resulted in a consistently high frequency of digital duplications, whereas anterior to posterior grafts produced very few. Similarly, dorsal to ventral grafts resulted in a good frequency of muscle duplications, whereas ventral to dorsal grafts did not. Such asymmetrical behavior is not predicted by most models involving local cell:cell interactions and the significance of the results for theories of pattern formation is discussed.

The regeneration of axolotl limbs covered by frog skin

Forearm skin of Stage XXIV Rana pipiens, which cannot regenerate limbs, was removed and placed upon the skinned forearms of young axolotls. The axolotl limbs were amputated immediately through the level of the grafts. Frog epidermis migrated to cover the amputation surface. Dedifferentiation and early blastema formation occurred beneath the frog wound epidermis. Limb regeneration continued, but in time axolotl epidermis overgrew the frog epidermis. The experiment shows that epidermis from nonregenerating frog limbs is still capable of supporting typical epimorphic regeneration.

Vitamin A and pattern formation in the regenerating limb.

Vitamin A and its derivatives, the retinoids, cause specific alterations in the proximo-distal pattern of regenerating axolotl limbs. In some cases complete limbs grow from carpal level amputations. If retinoids are responsible for changing the positional information of limb cells, these compounds may represent valuable probes for investigating the molecular basis of pattern formation.

Intercalary regeneration of symmetrical thighs in the axolotl, Ambystoma mexicanum

Intercalary regeneration of stylopodial and zeugopodial skeletal elements takes place in axolotl limbs composed of normal wrist blastemas autografted or homografted to double half-anterior or half-posterior thighs. Analysis of the morphological pattern of the skeleton and, in homografts, of pigmentation pattern, shows that the intercalated elements are derived from the host double half-thigh. Intercalary regeneration from double half-posterior thighs is expected since they normally can undergo complete proximal-distal regeneration, but is not necessarily expected from double half-anterior thighs, since they normally do not regenerate more distal segments. These results demonstrate that (1) cells of double half-anterior thighs are not inherently incapable of undergoing distal transformation, (2) cells of a distal blastema grafted to a more proximal level do not form patterns proximal to their level of origin, and (3) there is an inhibitory interaction between blastema cells derived from double half-anterior thighs that is expressed after simple amputation, but not when these cells are in contact with a more distal, normal blastema. Using these and other data, a three-dimensional boundary model of limb regeneration is proposed.

Segmental innervation of rotated and supernumerary axolotl hindlimbs.

The segmental nerve supply to axolotl limbs was misrouted by severing the limbs at the level of the femur, rotating them 180 degrees around their long axis, and then suturing them to the intact proximal stump. Following return of the blood supply to the rotated limb by the cross-anastomosing of blood vessels, a blastema often formed to the side of the rotation site giving rise to a supernumerary limb. The muscles of both rotated and supernumerary limbs were innervated by the segmental nerves. The percentage of cells innervated by segmental nerves 16 and 17 in each muscle was determined with intracellular electrodes at 14 weeks after the operation. Despite histological evidence that nerves had been misrouted in the rotated limb, the percentage innervation of each muscle by nerves 16 and 17 was similar to that in the unoperated contralateral controls. The same results were obtained for the supernumerary limbs. In some muscles a few synaptic sites were found innervated by segmental nerves which did not innervate that muscle in the contralateral controls. These had synaptic potentials with very low quantal contents if immediately adjacent sites were innervated by the segmental nerve, which did innervate that muscle in the contralateral controls. The results suggest that the selective properties of synaptic sites are alone sufficient to determine the entire segmental innervation pattern of the muscles in a limb.

Morphogenetic properties of the skin in axolotl limb regeneration

A study has been made of the morphogenetic properties of anterior and posterior skin from the lower forelimb of the axolotl. The basic experiment consisted of a graft of a half cuff of skin from a donor to a host limb followed by a 2-week healing period, amputation through the graft, and a study of the resulting regenerate. Limbs with double posterior skin formed double posterior regenerates and, in contrast, limbs with double anterior skin formed normal or slightly hypomorphic regenerates. Posterior skin from post-metamorphic animals had a similar but weaker effect to that from ordinary axolotls. Immunological rejection of allografts could be completely avoided if the donor limb was transplanted to the flank of the host when both were at the stage of tail-bud embryos, and the skin graft was later carried out between the supernumerary limb and one of the host limbs. This technique was used to show that immunological rejection does not affect the formation of duplicates from the limbs with double posterior skin, and to facilitate the studies of the cellular provenance of the regenerate. The cellular composition of duplicate regenerates was studied by using both triploid donors and triploid hosts. It was shown that the posterior side of the duplications consisted wholly of host tissue and the anterior side consisted of mixed donor and host tissue. Formation of the duplicated regenerate therefore seems to involve positional reprogramming of both donor and host tissues together with metaplasia of the donor tissue. It was not possible to inhibit the duplication-inducing property of posterior skin by treatment with a variety of enzymes. A model based on the serial threshold theory of regeneration is advanced to explain the results. This model successfully accounts for the observed non-equivalence of anterior and posterior skin, and also explains the different regeneration behaviour of anterior and posterior half limbs, the limited regeneration of double anterior limbs, and the pattern expansion and contraction shown by regenerates from double posterior limbs.

Experiments on developing limb buds of the axolotl Ambystoma mexicanum

Various experiments were performed on the limb buds of axolotls to compare the behaviour of amphibian limbs with that previously reported for chick limbs. Following removal of the tip or whole limb bud, extensive powers of regulation were observed since complete limbs always formed. Similarly after distal to proximal grafts intercalary regulation occurred to produce perfect limbs and after proximal to distal grafts serial repetitions resulted. Transplantation and rotation of limb buds to reverse either the dorso-ventral, antero-posterior or both axes resulted in the induction of supernumerary limbs in a large proportion of cases. Such regulatory behaviour of axolotl limb buds is in contrast to the mosaic nature of chick limbs and as a result, theories such as the progress-zone theory which have been formulated on the basis of data from chick limbs are not relevant to general principles of vertebrate limb development. Possible reasons for the diverse behaviour between the two systems are discussed.

Axon reflexes in axolotl limbs: Evidence that branched motor axons reinnervate muscles selectively

Numerous axon reflexes were found in grafted forelimbs, regenerated forelimbs, and forelimbs with regenerated nerves in the axolotl Ambystoma mexicanum. They were not seen in normal limbs. Each reflex was due to a single motor axon which branched, generally at a point central to the limb. In most cases several consecutive electrical stimuli were necessary to cause a visible contraction or a muscle action potential in the motor units concerned. I observed a total of 13 axon reflexes between the supernumerary limb and the ipsilateral host limb. All but one could be stimulated in either direction. In other animals I demonstrated 13 axon reflexes by cutting a small nerve in the distal limb and stimulating its proximal stump. In at least five, and possibly as many as 14 of these 26 axon reflexes, the two muscles involved were synonymous. In 20 of the 26, the muscles involved were situated in the same limb region and were synergistic or synonymous. Only in five axon reflexes were the muscles in widely different parts of the limb and clearly unrelated in function. Random innervation or mechanical guidance alone cannot account for these muscle-specific axon reflexes. Axon branches innervated muscles selectively, although the mechanism remains unclear. The selectivity was not perfect, though, because axon reflexes between dissimilar muscles did occur occasionally. Although it has been proposed that an axon may remain in an inappropriate muscle but be functionally “repressed,” no evidence of such repressed synapses was found.

Regenerative responses in larval axolotl limbs with skin grafts over the amputation surface

Complete flaps of whole skin over the amputation surface of larval axolotl limbs did not prevent dedifferentiation, DNA synthesis, and mitosis. However, even by two weeks, insufficient cell division had occurred for blastema formation. In about 50% of the cases, small areas of wound epidermis formed over the amputated tip of the radius or ulna. At two weeks after amputation, mitotic figures and dedifferentiated cells which incorporated H3-thymidine were predominantly located in the vicinity of the small area of wound epidermis. Although delayed in relation to controls, these limbs regenerated. The results are consistent with the view that the wound epidermis is necessary to maintain dedifferentiated cells in the cell cycle.

The development of synaptic connections between different segmental motoneurones and striated muscles in an axolotl limb

The pattern of innervation of the hindlimb flexor muscle surface of the mature axolotl by segmental nerves 16 and 17 (i.e., SN16 and 17) is approximately constant, if there is no innervation by nerve 18. If the proximal flexor muscle surface is divided into six equal sectors, it is found that sector 1 is only innervated by SN17, sector 2 by both SN16 and 17, sectors 3 and 4 by SN16, and sectors 5 and 6 by both SN16 and 17. In tadpole axolotls, when the flexor hindlimb muscle is about 1.5 mm long, muscle cells in all sectors can be found which were innervated by both SN16 and 17. During subsequent development the incorrect synaptic contacts are lost, and by the time the flexor muscle has reached a length of 5.5 mm the mature pattern of innervation is attained. At later stages of development a further loss of synapses is observed which appears to be unassociated with any change in the pattern of innervation of the flexor hindlimb muscle. These observations suggest the hypothesis that very early during development segmental motoneurones make incorrect connections on muscle cells, with the terminals of foreign motoneurones regressing in favour of the terminals of correct motoneurones.

Neurotrophic control of the cell cycle during amphibian limb regeneration

It is shown here that amputated and denervated limbs of larval axolotls dedifferentiate and a proportion of the cells released undergo DNA synthesis and mitosis. When the limb is denervated prior to amputation fewer cells go through the cell cycle, implying the existence of a pool of trophic factor in the limb. Recent work has demonstrated that denervated blastemal cells accumulate in the G1 phase of the cycle. These results strongly argue against the theory that the trophic factor controls the G2 phase. Rather, it is proposed that this factor regulated either the total number of cells cycling or the rate at which they cycle by varying the length of the G1 phase.

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.

The cell cycle during amphibian limb regeneration.

The duration of the cell cycle in the blastema of regenerating limbs of axolotls has been measured by means of [3H]thymidine pulse labelling and autoradiography. A chase was required to define the pulse period. An average cell cycle at 20 degrees C takes 53 h, S-phase takes 38 h; including parts of mitosis, G1 is 10 h and G2 is 5 h long. The protracted cycle and S-phase are consonant with the large genome in axolotis and other urodeles. The rapidly growing blastema probably contains a steady population of about 5000 proliferating cells, as there is a regular withdrawal of differentiating cells from the population. The kinds of determination which exist in this population of cells, or are exerted on it, are briefly considered.