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.     

Effects of retinoids on regenerating limbs: comparison of retinoic acid and arotinoid at different amputation levels

Summary Retinoic acid and the synthetic retinoid, arotinoid, were compared for their efficacy in inducing proximodistal (PD) pattern duplication in regenerating axolotl limbs, after amputation through either the distal zeugopodium (lower arm or leg) or distal stylopodium (upper arm or leg). At each level of amputation, the morphology of the duplications produced was the same for both retinoids, and the mean level of proximalization was dose-dependent. Blastema formation was delayed by both retinoids and the delay was associated with regression of the limb stump. Blastemas which produced PD duplication to the stylopodial or girdle level grew out from the stump in a posterior direction. In several zeugopodial regenerates, a partially duplicated, PD-reversed zeugopodium regenerated between the stump cartilages and a completely duplicated zeugopodium distally. Arotinoid was 50 times more effective than retinoic acid in evoking duplication. The dose of arotinoid required to duplicate a stylopodium in a stylopodial regenerate was several times higher than the dose required to duplicate a zeugopodium in a zeugopodial regenerate, suggesting differences either in the sensitivity of zeugopodial and stylopodial cells to retinoid, or in the numbers of positional value specifying these segments.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

Gallium nitrate: effects on cartilage during limb regeneration in the axolotl,Ambystoma mexicanum

Gallium nitrate, a drug shown to have efficacy in Paget's disease of bone, hypercalcemia of malignancy, and a variety of experimental autoimmune diseases, also inhibits the growth of some types of cancer. We examined dose and timing of administration of gallium nitrate on limb regeneration in the Mexican axolotl, Ambystoma mexicanum. Administered by intraperitoneal injection, gallium nitrate inhibited limb regeneration in a dose-dependent manner. Gallium nitrate initially suppressed epithelial wound healing and subsequently distorted both anterior-posterior and proximo-distal chondrogenic patterns. Gallium nitrate given at three days after amputation severely inhibited regeneration at high doses (6.25 mg/axolotl) and altered the normal patterning of the regenerates at low doses (3.75 mg/axolotl). Administration of 6.25 mg of gallium nitrate at four or 14 days prior to amputation also inhibited regeneration. In amputated limbs of gallium-treated axolotls, the chondrocytes were lost from inside the radius/ulna. Limbs that regenerated after gallium treatment was terminated showed blastema formation preferentially over the ulna. New cartilage of the regenerate often attached to the sides of the existing radius/ulna proximally into the stump and less so to the distal cut ends. J. Exp. Zool. 293:384-394, 2002.     

The relationship between migration and chondrogenic potential of trunk neural crest cells in Ambystoma mexicanum

Based on results of transplantation experiments, it has long been believed that trunk neural crest cells are incapable of chondrogenesis. When pigmented trunk neural crest cells of Ambystoma mexicanum are transplanted to cranial levels of albino (a/a) embryos, the graft cells ultimately produce ectopic fins, but are incapable of following the chondrogenic cranial neural crest pathways. Therefore, heterotopic transplantation does not expose these cells to the same environment experienced by cranial neural crest cells, and is neither an adequate nor a sufficient test of chondrogenic potential. However, in vitro culture of trunk neural crest cells with pharyngeal endoderm does provide a direct test of chondrogenic ability. That cartilage does not form under these conditions demonstrates conclusively that trunk neural crest cells possess no chondrogenic potential.     

Identification of pigment cells during early amphibian development (Triturus alpestris,Ambystoma mexicanum)

Summary The purpose of the present investigation was to provide and apply a methodological manual with which the distribution, patterning and relationship of melanophores and xanthophores can be analyzed during early amphibian development. For demonstration of the methods, which include ultrastructural, histochemical and biochemical approaches, Triturus alpestris and Ambystoma mexicanum (axolotl) embryos are used. These two species differ conspicuously in their larval pigment patterns, showing alternating melanophore bands in horizontal (T. alpestris) and vertical (axolotl) arrangements. With transmission- and scanning electron microscopy melanophores and xanthophores were distinguished by their different pigment organelles and surface structures. The presence of phenol oxidase (tyrosinase) was used to reveal externally invisible or faintly visible melanophores by applying an excess of 3,4 dihydroxy-phenylalanine (dopa). Xanthophores were made visible in fixed and living embryos by demonstrating their pterin fluorescence. In addition, pterins were analyzed by HPLC in embryos before and after pigmentation was visible.Abbreviations DOPA dihydroxy-phenylalanine - FCS fetal calf serum - FIF formaldehyde-induced fluorescence - FITC fluorescein isothiocyanate - HPLC high performance liquid chromatography Dedicated to the memory of Dr. Michael Claviez     

Xanthophores in chromatophore groups of the premigratory neural crest initiate the pigment pattern of the axolotl larva

Summary The barred pigment pattern (Lehman 1957) of the axolotl larva is best observed from stage 41 onwards, where it already consists of alternating transverse bands of melanophores and xanthophores along the dorsal side of the trunk. The present study investigateswhen the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined andhow they interact to create the barred pigment pattern. The presence of phenol oxidase (tyrosinase) in melanophores (revealed by dopa incubation) and pteridines in xanthophores (visualized by fluorescence) were used as markers for cell differentiation in order to recognize melanophores and xanthophores before they became externally visible. It was found that melanophores and xanthophores were already determined in the premigratory neural crest, at stages 30/31 and 35–36, respectively. Between stages 35–36 and 38 they were arranged in a prepattern of several distinct, mixed chromatophore groups along the dorsal trunk, morphologically correlated in the scanning electron microscope with humps on the original crest cell string. While the occurrence of xanthophores was restricted to the chromatophore groups and around them, melanophores were already uniformly distributed in the dorsolateral flank area, having migrated from trunk neural crest portions including the groups. The bar component of the pigment pattern was subsequently initiated by xanthophores, which caused melanophores in and around the chromatophore groups to fade or become invisible. The barred pattern was established by the formation of alternating clusters of like cells, melanophores and xanthophores.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the pancreas of the urodele amphibian,Ambystoma mexicanum

Summary The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely to the aldehyde-fuchsinophilic B cells of the endocrine pancreas. The reactive cell population was detected by use of one polyclonal antiserum against bovine insulin and eight different monoclonal antibodies against insulins from various mammalian species. Six of these antibody clones have known specificity to sub-regions of the insulin molecule. Additionally, fractions of an ethanol-HCl extract of pancreatic tissue from Ambystoma was studied in both conventional dot-blot tests by means of the same panel of antibodies and a two-site sandwich time-resolved immunofluorometric assay for human insulin involving two of the monoclonal antibodies. These experiments support the immunocytochemical observations by demonstrating the existence of an insulin-related peptide with a great deal of structural resemblance to mammalian insulins and displaying antigenic determinants in common at least with the amino acid residues A_8–10 and B_26–30. In conclusion, we interpret the findings as indicating that the immunocytochemically revealed tissue bound antigen in the Ambystoma pancreatic B-cells may be a peptide related to human insulin.Supported in part by SNF grant 11-5082 (G.N.H.). The authors are indebted to Dr. P. Rosenkilde for the gift of the Ambystoma material     

Retinoid antagonists inhibit normal patterning during limb regeneration in the axolotl, Ambystoma mexicanum

Retinoic acid (RA) has been detected in the regenerating limb of the axolotl, and exogenous RA can proximalize, posteriorize, and ventralize blastemal cells. Thus, RA may be an endogenous regulatory factor during limb regeneration. We have investigated whether endogenous retinoids are essential for patterning during axolotl (Ambystoma mexicanum) limb regeneration by using retinoid antagonists that bind to specific RAR (retinoic acid receptor) or RXR (retinoid X receptor) retinoid receptor subtypes. Retinoid antagonists (Ro41-5253, Ro61-8431, LE135, and LE540) were administered to regenerating limbs using implanted silastin blocks loaded with each antagonist. The skeletal pattern of regenerated limbs treated with Ro41-5253 or Ro61-8431 differed only slightly from control limbs. Treatment with LE135 inhibited limb regeneration, while treatment with LE540 allowed relatively normal limb regeneration. When LE135 and LE540 were implanted together, regeneration was not completely inhibited and a hand-like process regenerated. These results demonstrate that interfering with retinoid receptors can modify pattern in the regenerating limb indicating that endogenous retinoids are important during patterning of the regenerating limb.     

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

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

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

Pleiotropic effects of the Cardiac-lethal gene in the axolotl (Ambystoma mexicanum)

Embryos of the axolotl affected with the cardiac-lethal mutation form hearts that never begin to beat. A number of other traits characteristic of the mutant phenotype, including edema, underdeveloped gills, shorter stature, and aphagia (the inability to feed), were believed to be secondary effects of the absence of circulation. We have recently demonstrated that the pre-cardiac mesoderm is directly affected by the c gene, making it unresponsive to normal inductive signals. In this study, we replaced part or all of the mutant pre-cardiac mesoderm with wild-type tissue, to produce embryos with normally beating hearts and circulation. As expected, most of the other mutant characteristics were also corrected. However, otherwise normal individuals remained aphagic. All embryos with beating hearts containing mutant tissue also suffered from an unexpected circulatory arrest some time after the onset of circulation. This apparently indicates that there are at least two tissues other than the myocardium which appear to be directly affected by the c gene. These previously unsuspected pleiotropic effects of the mutation may involve poorly-characterized mesodermal-neural crest inductive interactions and may also lead to a greater understanding of the link between congenital heart defects and feeding difficulties in humans. © 1993Wiley-Liss, Inc.     

Three muscle fibre types in the axial muscle of axolotl (Ambystoma mexicanum Shaw) A quantitative lightand electron microscopic study

Morphometric analysis by light microscopy of p-phenylene-diamine stained semithin sections of axolotl tail muscle revealed differences in the cross-sectional area of the fibres and in the number of mitochondria and of lipid inclusions per fibre, and indicated the presence of three distinct types of fibres. The tripartition was found to be statistically highly significant. Representative fibres from each group established by light microscopic morphometry were subjected to an ultrastructural morphometric analysis. The volume content of mitochondria amounted to 9.8% of the fibre volume for red, 4.0% for intermediate and 0.8% for white fibres. The myofibrils composed 60%, 70% and 83% in the same fibres. The volume of the sarcotubular system (t-tubuli and sarcoplasmic reticulum) was 2.5% in red, 4.5% in intermediate and 11.7% in white fibres. The three fibre types also demonstrated differences in myofibrillar cross-striation pattern and number of triads. The reliability of the light microscopic morphometry was tested by correlation with EM montages of the representative fibres.     

Serially duplicated regenerates from the anterior half of the axolotl limb after retinoic acid treatment

Summary Axolotl (Ambystoma mexicanum) forearms were divided, by an incision between the radius and ulna, to produce anterior and posterior halves. These were prevented from fusing together again by a graft of head skin and amputated through the wrist. This procedure enabled independent regeneration from both halves of the stump. Anterior half stumps produced a single digit while the posterior halves mainly regenerated three digits, the two halves together making a single hand. Treatment with retinoic acid, injected intraperitoneally four days after amputation, abolished regeneration from the posterior half stump and produced proximo-distally duplicated regenerates from the anterior half. The duplicated regenerates had in most cases a complete four digit hand and were therefore more than proximalised regenerates from the anterior side of the limb. Replacement of anterior limb skin with head skin had no effect on the response of the regenerating limb to retinoic acid. In species where application of retinoic acid induces anterior-posterior duplications, these are always derived from the anterior side of the limb. The results presented here show that the morphogenic effects of retinoic acid in inducing proximo-distal duplications are also due to its effects on the anterior tissues of the limb.Excellent technical assistance was provided by Carole Ross and Marjory Shiach and useful discussion were had with Paul Martin, David Wilson and Gavin Swanson     

The axolotl thymus: cell types of the microenvironment

Summary The three-dimensional structure of the axolotl (urodele amphibian) thymus was studied by combined scanning and transmission electron microscopy. The epithelial cell is the major component of the microenvironment forming the meshwork where thymocytes differentiate. Three different types of epithelial cells could be defined by their intracytoplasmic organelles and their localization in the subcapsular or deeper part of the organ. These epithelial cells participate in various types of lymphostromal interactions. Other stromal elements, such as interdigitating reticular cells, macrophages, eosinophil granulocytes and epithelial cysts were also defined. The absence of a true cortico-medullary differentiation in the axolotl thymus, the presence of different stromal elements and the physiological significance of these various microenvironments are discussed.     

Axial characteristics of nerve induced supernumerary limbs in the axolotl

Summary Supernumerary limbs were produced by deviating the sciatic nerve to the surface of the axolotl hindlimb either alone or in combination with small skin grafts from specific limb positions. With no skin grafts a very low frequency of good supernumeraries were produced. However, when associated with skin grafts, this frequency was significantly increased. The pattern of skeletal elements and muscles were analysed in the supernumeraries which formed at each location. In both the anterior-posterior and dorsal-ventral axes specific anatomical features were found which correlated with their position of origin on the host limb. Characteristic features were also observed with respect to the proximal-distal axis of the outgrowths. These phenomena are discussed in relation to our current understanding of the rules of pattern regulation in the regenerating limb.     

Effect of marginal vitamin A deficiency during pregnancy on retinoic acid receptors and N-methyl-D-aspartate receptor expression in the offspring of rats

This study examined whether pregnancy-related marginal vitamin A deficiency (MVAD) influences postnatal development of retinoic acid receptors (RARs) and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) in hippocampus of rat pups. Sixteen female rats were randomized equally into control and MVAD groups. Dams and pups were fed with either a normal control diet or one deficient in vitamin A. Eight female pups in each group were killed at 1 day, 2 weeks, 4 weeks and 8 weeks after birth, respectively. Serum retinol levels were monitored. The messenger RNA (mRNA) and protein expressions and subcellular localization of RARα, RARβ and NR1 in postnatal hippocampus were detected. At 1 day, 2 weeks and 8 weeks after birth, serum retinol levels in the MVAD group were significantly lower than those in the control group. Results of Morris water maze test at 7 weeks of age showed that spatial learning and memory in the MVAD group were affected. Vitamin A deficiency resulted in decreased mRNA levels of RARα, RARβ and NR1 (P<.05). The protein level of RARα and NR1 in the MVAD group was lower than that of the control group (P<.05). There was no significant difference in RARβ between the groups (P>.05). A mass of RARα and NR1 colocalized in hippocampal cell cytoplasm on postnatal day 1. Our data suggested that vitamin A deficiency in pregnancy may affect the postnatal expression of RARα and NR1, affecting learning and memory function in the hippocampus and synaptic plasticity of the calcium signaling pathway.