Regeneration of symmetrical forelimbs in the axolotl,Ambystoma mexicanum

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978).     

Morphogenetic interactions occurring between blastemas and stumps after exchanging blastemas between normal and double-half forelimbs in the axolotl, Ambystoma mexicanum.

Regeneration blastemas were exchanged between surgically constructed forelimbs comprised of symmetrical tissues (double-anterior and double-posterior) and normal, unoperated forelimbs. Normal blastemas grafted at the stage of medium bud (MB) onto double-half forelimb stumps regenerated normal skeletal patterns in nearly all cases. Double-half blastemas transplanted at the stage of MB onto normal forelimb stumps did not regenerate complete limb patterns. These results indicate that a double-half blastema cannot be “rescued” by transplantation to a normal stump and that a double-half limb stump does not interfere with the ability of a normal blastema to distally transform. The regeneration blastema possesses sufficient positional information at the stage of MB to permit it to develop autonomously. Supernumerary forelimbs resulted from several types of graft-stump combinations. The location and handedness of these supernumerary limbs are predicted by the rules of a recently presented model for pattern regulation in epimorphic fields [French, V., Bryant, P. J., and Bryant, S. V. (1976). Science193, 969–981].     

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.     

Resegmentation in the mexican axolotl,Ambystoma mexicanum

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well‐studied using the quail–chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups. J. Morphol. 275:141–152, 2014. © 2013 Wiley Periodicals, Inc.     

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

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

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.     

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.     

An introduction to the Mexican axolotl (Ambystoma mexicanum)

A number of unusual traits, including a remarkable capacity for wound healing and limb regeneration, make the axolotl an interesting animal model. The author provides an overview of axolotl care and use in biomedical research.     

The occurrence of supernumerary limbs following blastemal transplantation in the regenerating forelimb of the axolotl, Ambystoma mexicanum

Regeneration blastemas at the stages of medium bud and palette were transplanted to contralateral limb stumps so that either their anterior and posterior positions or their dorsal and ventral positions were apposed to those of the stumps. Grafts were shifted from distal levels to proximal levels, or from proximal levels to distal levels, or remained at either a proximal or a distal level. When anterior and posterior positions of graft and stump were apposed, supernumerary limbs were produced at the graft-stump junction in anterior and posterior positions relative to the stump. All analyzable supernumerary limbs were of stump handedness. Apposition of dorsal and ventral positions of graft and stump led to the formation of supernumerary limbs at dorsal and ventral positions relative to stump tissues. All analyzable supernumerary limbs were once again of stump handedness. Shifts from distal levels to proximal levels never resulted in skeletal deletions, as potential deletions in the proximal-distal axis were always filled in. Shifts from proximal levels to distal levels resulted in a low frequency of serial duplications. The results are discussed in view of a recently presented formal model for pattern regulation in epimorphic fields.     

Development of the sympathetic system in the Mexican axolotl, Ambystoma mexicanum

Migration of trunk neural crest cells in axolotl embryos has been followed by autoradiography using grafts of [³H]thymidine-labeled neural folds. Crest cells form melanocytes, dorsal fin mesenchymal cells, spinal ganglion cells, and reach the sympathetic region. Sympathetic neurons, however, are not identifiable morphologically until about 6 weeks posthatching, in 24-mm larvae. At this stage, neurons, although few, have characteristic ultrastructure and receive synapses. The diffuse ganglia also contain innervated chromaffin cells; these differentiate earlier, a few days posthatching, in 14-mm larvae. A third type of cell is of morphologically indifferent appearance. Catecholamine-specific formaldehyde-induced fluorescence first appears clearly at 14 mm; with growth, the number of fluorescent cells increases. Series of larvae were injected intraperitoneally with nerve growth factor (NGF), six 30-unit injections over 2 weeks. NGF treatment increases the number of neurons apparent in 24-mm larvae. Furthermore, differentiated neurons occur in NGF-treated 20-mm larvae (about 4 weeks posthatching), when there are none in controls. The early appearance of differentiated chromaffin cells and the relatively late appearance of differentiated sympathetic neurons suggest that adrenergic functions during the first few weeks of larval life are controlled humorally by the chromaffin cells, and that at 24 mm, neurons begin to provide faster, finer control.     

The surfactant system of the lung in the Mexican axolotl Ambystoma mexicanum

The broncho-alveolar space of axolotl contains numerous osmiophilic structures, which have been classified morphologically into 3 types of inclusions. Type I inclusions exhibited lattices of square to rectangular grid patterns with membranous elements 6 nm thick. This lattice was crossed by a 2 nm dense line. Type II inclusions were composed of 7 nm dark lines and 15.5 nm light lines in an alternating repeating pattern. Furthermore, the light lines showed an intrapenoid line of 2 nm. Type III inclusions consisted of concentric whorls of lamellae similar in form to spider webs. Only type III inclusions were identified in grand alveolar cells.     

A staging system for forelimb regeneration in the axolotl,Ambystoma mexicanum

A staging system has been devised for normal regeneration from the upper arm in the mature axolotl. It consists of seven externally definable stages: (1) Wound healing (WH); (2) Dedifferentiation (DD); (3) Early bud (EB); (4) Medium bud (MB); (5) Late bud (LB); (6) Palette (Pal), and (7) Digital outgrowth (DO). Serial histological sections of 38 regenerating limbs were used to correlate gross stages with microscopic events in the regenerative process.     

The timing of morphogenetic events in the regenerating forelimb of the axolotl,Ambystoma mexicanum

The timing of morphogenetic events in the regenerating forelimb of the axolotl was investigated by rotation of limb coverings at well-defined stages in the regenerative process. Both the skin covering the stump and the epidermis covering the regenerate were manipulated independently and together as a unit. The results show that the transmission of morphogenetic information covers a broad range of regenerative stages. This morphogenetic information seems first to become irreversibly fixed in the regenerate by the stage of late bud. The regenerate is sensitive to stump influences at early stages of regeneration, but it becomes insensitive to stump influences by the stage of palette. Evidence is presented which implies that epidermis that covers the regenerate is capable of influencing morphogenesis.     

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.     

Activity of lateral line efferents in the axolotl (Ambystoma mexicanum)

Summary Activity of efferent fibers was recorded from the ramus ophthalmicus superficialis of the head lateral line nerve and the ramus medialis of the trunk lateral line nerve of the axolotl Ambystoma mexicanum. Baseline activity and activity evoked by sensory stimuli were examined. Electrical stimulation of selected branches was used to determine the conduction velocity and the branching pattern of efferent fibers. The influence of lesions at different levels in the CNS on efferent activity was studied.Up to 5 units with baseline activity were found in a single ramus of the lateral line nerve. Discharge rates were variable and highly irregular; they differed between units of the same branch. Bursting activity occurred in 62% of the units. Movements of the animal were accompanied by activity in up to 8 efferent units in a single nerve.Efferent activity could be elicited or modified by stimulation of visual, labyrinthine, somatosensory, and lateral line systems. Stimulation of the electrosensory system had no effect. Individual efferent neurons innervated different fields in the lateral line periphery. Conduction velocities of efferent fibers ranged from 5 to 12 m/s.Efferent units received input from various sources at different brain levels up to the diencephalon. These in puts determined the baseline activity. The mechanosensory input was mediated at the medullary level.Abbreviations r.m. ramus medialis - r.o.s. ramus ophthalmicus superficialis - r.s. ramus superior     

Isolation of Mhc class I cDNAs from the axolotl Ambystoma mexicanum

 Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Iα molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of β₂-microglobulin with the Mhc α chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the α1 and α2 domains suggests the classical nature of axolotl class I A genes. Received: 3 June 1996 / Revised: 14 October 1996     

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.