Ultrasound description of the coelomic cavity of the axolotl (Ambystoma mexicanum) in a clinically healthy population: a pilot study.

Axolotls (Ambystoma mexicanum) are extensively studied for their relevance in human medical research. Despite being critically endangered in the wild, they have gained popularity as household pets. Although they have been kept in captivity for over a century, detailed descriptions of their coelomic organ anatomy remain limited. Also, this species exhibits significant variations compared to other amphibians. Ultrasound is a non-invasive and painless medical imaging technique, ideally suited for investigating internal organs or structures. This study focused on describing the ultrasound appearance of the axolotl coelomic cavity. It details the identification, localization and parenchymal description of major organs in 28 neotenic axolotls using ultrasound frequencies ranging from 7 to 15 MHz. The accuracy of the results was validated by comparing ultrasound findings with necropsy results from one male and one female axolotl. The heart, lung surface, liver and reproductive tracts were visualized. Measurements, along with confidence intervals, were calculated for the spleen, kidneys, testicles, gastric wall, gallbladder, and pylorus. Occasional detection of hyperechoic millimetric particles in the gallbladder or ascites was noted. However, visualization of the pancreas and bladder was not possible. This research outcomes involve the development of a comprehensive atlas comprising images obtained throughout the study. Additionally, the experiment established a reproducible and readily accessible protocol for conducting anatomy-morphological assessments in axolotl medicine. This protocol stands as a crucial preliminary stage before advancing to lesion identification.

Descripción morfométrica de la anatomía externa e interna del Ambystoma mexicanum / Morphometric description of the external and internal anatomy of Ambystoma mexicanum

RESUMEN: El Ambystoma mexicanum o comúnmente llamado axolote, es un anfibio miembro del género Ambystoma que comprende 32 especies presentes desde el sur de Canadá hasta la región central de México. Actualmente esta especie se encuentra en peligro de extinción debido a cambios fisicoquímicos en su medio ambiente y la depredación por organismos introducidos en su hábitat. Dentro de las múltiples estrategias para su conservación se encuentran el desarrollo de programas de investigación, educación y desarrollo del manejo clínico. El objetivo del presente trabajo estuvo enfocado en obtener y analizar datos morfométricos acompañados por una descripción de la anatomía externa e interna de la especie Ambystoma mexicanum con el propósito de contribuir a su conservación. Por lo anterior, cinco ejemplares de la especie Ambystoma mexicanum de 2 años, criados en cautiverio, fueron estudiados para obtener valores morfométricos externos e internos utilizando un calibrador vernier, balanzas analíticas y rayos X. Los resultados de la observación externa mostraron el dimorfismo sexual característico de estas especies presentes en la zona peri cloacal, además, el análisis radiográfico permitió observar y señalar algunas estructuras óseas del esqueleto axial y apendicular. Los ejemplares presentaron un peso promedio de 31,6 g y una longitud de 15,7 cm. Los miembros anteriores mostraron una longitud de 2,92 cm y 2,8 cm en los miembros posteriores. El análisis de los órganos internos mostró que el corazón tiene un peso de 0,036 g y una longitud de 0,75 cm, los pulmones un peso de 0,019 g y una longitud de 2,6 cm, el estómago arrojó una longitud de 4,86 cm y el intestino 10,88 cm. En conclusión, los valores presentados en el presente trabajo sirven de referencia en futuros trabajos de investigación clínica veterinaria y manejo zootécnico del axolote para su conservación y preservación.

SUMMARY: The Ambystoma mexicanum commonly known as axolotl, is an amphibian and member of the genus Ambystoma which includes 32 species that can be found from southern Canada to central Mexico. Currently this species is in danger of extinction due to physicochemical changes in its environment and predation by organisms introduced into its habitat. Among the multiple strategies to aid in its conservation are the development of research programs, education and development of clinical management. The objective of this work is focused on obtaining and analyzing morphometric data accompanied by a description of the external and internal anatomy of the species Ambystoma mexicanum with the purpose of contributing to the conservation of this species. Therefore, five 2-year-old specimens of the Ambystoma mexicanum species, raised in captivity, were studied to obtain external and internal morphometric values using a vernier caliper, analytical balances and X-rays. The results of external observation showed the characteristic sexual dimorphism of these species present in the pericloacal zone. Furthermore, the radiographic analysis allowed to observe and point out some bony structures of the axial and appendicular skeleton. The specimens presented an average weight of 31.60 g and a length of 15.70 cm. The forelimbs showed a length of 2.92 cm and 2.8 cm in the hindlimbs. Analysis of the internal organs showed that the heart had a weight of 0.036 g and a length of 0.75 cm, the lungs a weight of 0.019 g and a length of 2.6 cm, the stomach had a length of 4.86 cm and the intestine 10.88 cm. In conclusion, the values presented in this work serve as a reference for future veterinary clinical research and zootechnical management of the axolotl for its conservation and preservation.

MRI- and histologically derived neuroanatomical atlas of the Ambystoma mexicanum (axolotl).

Amphibians are an important vertebrate model system to understand anatomy, genetics and physiology. Importantly, the brain and spinal cord of adult urodels (salamanders) have an incredible regeneration capacity, contrary to anurans (frogs) and the rest of adult vertebrates. Among these amphibians, the axolotl (Ambystoma mexicanum) has gained most attention because of the surge in the understanding of central nervous system (CNS) regeneration and the recent sequencing of its whole genome. However, a complete comprehension of the brain anatomy is not available. In the present study we created a magnetic resonance imaging (MRI) atlas of the in vivo neuroanatomy of the juvenile axolotl brain. This is the first MRI atlas for this species and includes three levels: (1) 82 regions of interest (ROIs) and a version with 64 ROIs; (2) a division of the brain according to the embryological origin of the neural tube, and (3) left and right hemispheres. Additionally, we localized the myelin rich regions of the juvenile brain. The atlas, the template that the atlas was derived from, and a masking file, can be found on Zenodo at https://doi.org/10.5281/zenodo.4595016 . This MRI brain atlas aims to be an important tool for future research of the axolotl brain and that of other amphibians.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl's blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

A comparative study of fixatives for axolotl blastema tissue / Estudio comparativo de fijadores para el tejido blastema del axolotl

Regeneration is defined as tissue renewal and functional restoration process of the damaged parts of the body after an injury. Ambystoma mexicanum, commonly named the Axolotl, is one of the unique vertebrates, which has a remarkable ability to regenerate their extremities following the amputation. Although the process of regeneration includes several periods, it can be divided into two main phases; blastema formation and dedifferentiation. In the couple of hours following the amputation, wound closure occurs by migration of epithelial cells around the amputation site followed by macrophage infiltration and dedifferentiation of cells to turn into stem cells. Accumulated stem cells form a very authentic tissue type called blastema, which is crucial for successful regeneration. In order to evaluate this exceptional tissue and acquire high quality images, it is crucial to employ specific procedures to prepare the tissue for imaging. Here, in this study, we aimed to investigate success of various fixative solutions (Carnoy's, Bouin's, % 10 NBF, Clarke's, Alcoholic Formaline and AFA) to monitor the fixed blastema. Our data reveals that integrity of the blastema tissue differs among used fixatives and a significant difference is observed between the samples in terms of staining quality.

La regeneración se define como la renovación del tejido y el proceso de restauración funcional de las partes dañadas del cuerpo después de una lesión. Ambystoma mexicanum, comúnmente llamado Axolotl, es uno de los únicos vertebrados que tiene una notable capacidad para regenerar sus miembros después de una amputación. Aunque el proceso de regeneración incluye varios períodos, se puede dividir en dos fases principales: formación del blastema y desdiferenciación. En el par de horas después de la amputación, el cierre de la herida ocurre por la migración de células epiteliales alrededor del sitio de la amputación seguido por una infiltración de macrófagos y la desdiferenciación de las células para convertirse en células madre. Las células madre acumuladas forman un tipo de tejido muy diferenciado denominado blastema, que es crucial para una exitosa regeneración. Para evaluar este tejido y adquirir imágenes de alta calidad, es crucial emplear procedimientos específicos para la obtención de imágenes. En este estudio, se intentó investigar el éxito de varias soluciones fijadoras (Carnoy, Bouin, % 10 NBF, Clarke, Formalina Alcohólica y AFA) para monitorear la fijación del blastema. Nuestros datos revelan que la integridad del tejido del blastema difiere entre los fijadores utilizados y una diferencia significativa observada entre las muestras se da en términos de la calidad de tinción.

Localization of amylin-like immunoreactivity in melanocyte-stimulating hormone-containing cells of the pars intermedia but not those of the pars distalis in the axolotl (Ambystoma mexicanum) pituitary.

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactivity in the axolotl (Ambystoma mexicanum) pituitary. Amylin-immunoreactive cells were observed in the pars intermedia, and these cells were found to be immunoreactive for α-melanocyte-stimulating hormone (αMSH) as well. In contrast, αMSH-immunoreactive cells in the pars distalis were immuno-negaitive for amylin. These light microscopic findings were confirmed by immunoelectron microscopy. Amylin-immunoreactive signals were located on the haloes of presumable secretory granules in association with αMSH-immunoreactive signals in the amylin-positive cells. However, in the pars distalis, the αMSH-positive cells did not contain amylin-immunoreactive secretory granules. Western blot analysis of axolotl pituitary extracts revealed the labeling of a protein band at approximately 10.5-kDa by the anti-rat amylin serum, which was not labeled by the anti-αMSH antibody. These findings indicate that amylin secreted from MSH-producing cells in the pars intermedia may modulate MSH secretion in an autocrine fashion and may participate in MSH functions such as fatty homeostasis together with MSH.

Linking vertebral number to performance of aquatic escape responses in the axolotl (Ambystoma mexicanum).

Environmental conditions during early development in ectothermic vertebrates can lead to variation in vertebral number among individuals of the same species. It is often seen that individuals of a species raised at cooler temperatures have more vertebrae than individuals raised at warmer temperatures, although the functional consequences of this variation in vertebral number on swimming performance are relatively unclear. To investigate this relationship, we tested how vertebral number in axolotls (Ambystoma mexicanum) affected performance of aquatic escape responses (C-starts). Axolotls were reared at four temperatures (12-24°C) encompassing their natural thermal range and then transitioned to a mean temperature (18°C) three months before C-starts were recorded. Our results showed variation in vertebral number, but that variation was not significantly affected by developmental temperature. C-start performance among axolotls was significantly correlated with caudal vertebral number, and individuals with more caudal vertebrae were able to achieve greater curvature more quickly during their responses than individuals with fewer vertebrae. However, our results show that these individuals did not achieve greater displacements or velocities, and that developmental temperature did not have any effect on C-start performance. We highlight that the most important aspects of escape swim performance (i.e., how far individuals get from a threat and how quickly they move the most important parts of the body away from that threat) are consistent across individuals regardless of developmental temperature and morphological variation.

Regeneration of limb joints in the axolotl (Ambystoma mexicanum).

In spite of numerous investigations of regenerating salamander limbs, little attention has been paid to the details of how joints are reformed. An understanding of the process and mechanisms of joint regeneration in this model system for tetrapod limb regeneration would provide insights into developing novel therapies for inducing joint regeneration in humans. To this end, we have used the axolotl (Mexican Salamander) model of limb regeneration to describe the morphology and the expression patterns of marker genes during joint regeneration in response to limb amputation. These data are consistent with the hypothesis that the mechanisms of joint formation whether it be development or regeneration are conserved. We also have determined that defects in the epiphyseal region of both forelimbs and hind limbs in the axolotl are regenerated only when the defect is small. As is the case with defects in the diaphysis, there is a critical size above which the endogenous regenerative response is not sufficient to regenerate the joint. This non-regenerative response in an animal that has the ability to regenerate perfectly provides the opportunity to screen for the signaling pathways to induce regeneration of articular cartilage and joints.

Muscle development and differentiation in the urodele Ambystoma mexicanum.

Muscle differentiation has been widely described in zebrafish and Xenopus, but nothing is known about this process in amphibian urodeles. Both anatomical features and locomotor activity in urodeles are known to show intermediate features between fish and anurans. Therefore, a better understanding of myogenesis in urodeles could be useful to clarify the evolutionary changes that led to the formation of skeletal muscle in the trunk of land vertebrates. We report here a detailed morphological and molecular investigation on several embryonic stages of Ambystoma mexicanum and show that the first differentiating muscle fibers are the slow ones, originating from a myoblast population initially localized close to the notochord that forms a superficial layer on the somitic surface afterwards. Subsequently, fast fibers differentiation ensues. We also identified and cloned A. mexicanum Myf5 as a muscle-specific transcriptional factor likely involved in urodele muscle differentiation.

Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.

Glucose transporter distribution in the vessels of the central nervous system of the axolotl Ambystoma mexicanum (Urodela: Ambystomatidae).

The GLUT-1 isoform of the glucose transporter is commonly considered a reliable molecular marker of blood-brain barrier endothelia in the neural vasculature organized in a three-dimensional network of single vessels. The central nervous system of the axolotl Ambystoma mexicanum is characterized by a vascular architecture that contains both single and paired vessels. The presence and distribution of the GLUT-1 transporter are studied in this urodele using both immunoperoxidase histochemistry and immunogold technique. Light microscopy reveals immunopositivity in both parenchymal and meningeal vessels. The transverse-sectioned pairs of vessels do not show the same size. Furthermore, in the same pair, the two elements often differ in diameter. The main regions of the central nervous system show a different percentage of the paired structures. Only immunogold cytochemistry reveals different staining intensity in the two adjoined elements of a vascular pair. Colloidal gold particles show an asymmetric distribution in the endothelia of both single and paired vessels. These particles are more numerous on the abluminal surface than on the luminal one. The particle density is calculated in both vascular types. The different values could indicate functional differences between single and paired vessels and between the two adjoined elements of a pair, regarding glucose transport.

Differential expression of tropomyosin during segmental heart development in Mexican axolotl.

The Mexican axolotl, Ambystoma mexicanum, serves as an intriguing model to investigate myofibril organization and heart development in vertebrates. The axolotl has a homozygous recessive cardiac lethal gene "c" which causes a failure of ventricular myofibril formation and contraction. However, the conus of the heart beats, and has organized myofibrils. Tropomyosin (TM), an essential component of the thin filament, has three known striated muscle isoforms (TPM1alpha, TPM1kappa, and TPM4alpha) in axolotl hearts. However, it is not known whether there are differential expression patterns of these tropomyosin isoforms in various segments of the heart. Also, it is not understood whether these isoforms contribute to myofibril formation in a segment-specific manner. In this study, we have utilized anti-sense oligonucleotides to separately knockdown post-transcriptional expression of TPM1alpha and TPM4alpha. We then evaluated the organization of myofibrils in the conus and ventricle of normal and cardiac mutant hearts using immunohistochemical techniques. We determined that the TPM1alpha isoform, a product of the TPM1 gene, was essential for myofibrillogenesis in the conus, whereas TPM4alpha, the striated muscle isoform of the TPM4 gene, was essential for myofibrillogenesis in the ventricle. Our results support the segmental theory of vertebrate heart development.

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.

Role of cranial neural crest cells in visceral arch muscle positioning and morphogenesis in the Mexican axolotl, Ambystoma mexicanum.

The role of cranial neural crest cells in the formation of visceral arch musculature was investigated in the Mexican axolotl, Ambystoma mexicanum. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate) labeling and green fluorescent protein (GFP) mRNA injections combined with unilateral transplantations of neural folds showed that neural crest cells contribute to the connective tissues but not the myofibers of developing visceral arch muscles in the mandibular, hyoid, and branchial arches. Extirpations of individual cranial neural crest streams demonstrated that neural crest cells are necessary for correct morphogenesis of visceral arch muscles. These do, however, initially develop in their proper positions also in the absence of cranial neural crest. Visceral arch muscles forming in the absence of neural crest cells start to differentiate at their origins but fail to extend toward their insertions and may have a frayed appearance. Our data indicate that visceral arch muscle positioning is controlled by factors that do not have a neural crest origin. We suggest that the cranial neural crest-derived connective tissues provide directional guidance important for the proper extension of the cranial muscles and the subsequent attachment to the insertion on the correct cartilage. In a comparative context, our data from the Mexican axolotl support the view that the cranial neural crest plays a fundamental role in the development of not only the skeleton of the vertebrate head but also in the morphogenesis of the cranial muscles and that this might be a primitive feature of cranial development in vertebrates.

Patterns of spatial and temporal visceral arch muscle development in the Mexican axolotl (Ambystoma mexicanum).

Vertebrate head development is a classical topic that has received renewed attention during the last decade. Most reports use one of a few model organisms (chicken, mouse, zebrafish) and have focused on molecular mechanisms and the role of the neural crest, while cranial muscle development has received less attention. Here we describe cranial muscle differentiation and morphogenesis in the Mexican axolotl, Ambystoma mexicanum. To determine the onset of differentiation we use antibodies against desmin and optical sectioning using confocal laser scanning microscopy on whole-mount immunostained embryos. This technique makes it possible to document the cranial muscle in three dimensions while keeping the specimens intact. Desmin expression starts almost simultaneously in the first, second, and third visceral arch muscles (as in other amphibians studied). Muscle anlagen divide up early into the different elements which constitute the larval cranial musculature. We extend and refine earlier findings, e.g., by documenting a clear division between interhyoideus and interhyoideus posterior. The timing of cranial muscle differentiation differs among vertebrate groups, but seems to be constant within each group. This study provides a morphological foundation for further studies of muscle cell fate and early differentiation.

Discrimination of conspecific sex and reproductive condition using chemical cues in axolotls ( Ambystoma mexicanum).

Chemosensory cues play an important role in the daily lives of salamanders, mediating foraging, conspecific recognition, and territorial advertising. We investigated the behavioral effects of conspecific whole-body odorants in axolotls, Ambystoma mexicanum, a salamander species that is fully aquatic. We found that males increased general activity when exposed to female odorants, but that activity levels in females were not affected by conspecific odorants. Although males showed no difference in courtship displays across testing conditions, females performed courtship displays only in response to male odorants. We also found that electro-olfactogram responses from the olfactory and vomeronasal epithelia were larger in response to whole-body odorants from the opposite sex than from the same sex. In males, odorants from gravid and recently spawned females evoked different electro-olfactogram responses at some locations in the olfactory and vomeronasal epithelia; in general, however, few consistent differences between the olfactory and vomeronasal epithelia were observed. Finally, post hoc analyses indicate that experience with opposite-sex conspecifics affects some behavioral and electrophysiological responses. Overall, our data indicate that chemical cues from conspecifics affect general activity and courtship behavior in axolotls, and that both the olfactory and vomeronasal systems may be involved in discriminating the sex and reproductive condition of conspecifics.

Ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum with reference to ionic transport.

The ultrastructure of the external gill epithelium of the axolotl, Ambystoma mexicanum, has been examined using conventional transmission electron microscopy to elucidate its role in ionic transport. Four cell types are identified in the gill filament and primary gill bar epithelium. These are granular, ciliated, Leydig and basal cells. A fifth cell type, the flat mitochondria-rich cell is only found in the gill bar epithelium. The predominant granular cells display microvilli at their surface and their cytoplasm contains abundant mitochondria, rough endoplasmic reticulum, Golgi complexes, vesicles and PAS+ secretory granules that are extruded at the surface, which along with secretions from the Leydig cells form a mucous coat. The granular cells are joined apically by junctional complexes consisting of zonulae occludens, zonulae adherens and desmosomes. The lateral membranes of granular cells enclose large intercellular spaces that are closed at the apical ends but remain open at the basal ends adjoining capillaries. In AgNO3-treated axolotl, the gills become darkly stained, the silver grains penetrate apical membranes and appear in the cytoplasm, accumulating near the lateral membranes and also enter the intercellular spaces. These findings are consistent with the dual role of the gill epithelium in mucus production and active ionic transport.

The odontoclasts of Ambystoma mexicanum.

The resorption of teeth in Ambystoma mexicanum during postembryonal ontogenesis and induced metamorphosis occurs by means of light-microscopic detectable giant-cells. These have morphological and functional characters similar to those of odontoclasts of other vertebrates. The multinucleated odontoclasts resorb not only the pedicel (base), but the stalk of the tooth, too. When active, the cells form a ruffled border and a sealing zone. In this way they are able to demineralize the hard tissues of teeth (dentin and mineral of the pedicel) and to dissolve the extracellular matrix. Resorption of enamel has not been observed. Marks of resorption resemble the Howship's lacunae of other tetrapods. TRAP as a typical enzyme of odontoclasts could not be detected histochemically. Dependence of PTH, which is supposed to be necessary for the formation and activation of odontoclasts as well as of thyroxine can be excluded, although the resorbing cells are functionally and cytologically identical with those of other vertebrates. This demands some other mechanism for the formation and regulation of the odontoclasts in A. mexicanum.

The epithelium of the tongue of Ambystoma mexicanum. Ultrastructural and histochemical aspects.

The distribution pattern of taste buds and goblet cells and histochemical and ultrastructural aspects of the tongue epithelium of Ambystoma mexicanum are here described. This study is also concerned with the developmental stages and origins of the epithelial cells. Pavement cells and goblet cells of the stratum superficiale are replaced by basal cells of the stratum germinativum in larvae and neotenous adults. The pavement cells of the larvae are characterized by a marginal layer of mucin grana. Decompaction of the mucins occurs immediately before extrusion in the adult. The larval goblet cell type (type I), which is also present in the adult, contains unfused grana of irregular shape. At the tip of the tongue, a further type (type II) of goblet cells is found. In the type II cells the intracellular secretory grana fuse to a single homogeneous mass. Leydig cells of the tongue epithelium are discerned by light microscopy first in the semi-adult, apparently correlated with partial metamorphosis. In the course of ontogenesis and induced metamorphosis the secretion changes to neutral glycoconjugates. The mucins of the pavement cells change first followed by those of the goblet cells. The glands of the secondary tongue show a dorso-ventral pattern of varying secretory qualities. Taste buds are found at the anterior margin of the tongue and along the base of the gill clasps in the early larva. They are already distributed all over the tongue at the end of the early larval phase.

Hard tissue of teeth and their calcium and phosphate content in Ambystoma mexicanum (Urodela: Ambystomatidae).

The wall of the pulp cavity, fracture faces and the demineralized surfaces of teeth from larvae and adults of Ambystoma mexicanum were investigated by scanning electron microscopy (SEM). Calcium and phosphate contents were determined by microanalysis. The apical part of the tooth (crown, tooth apex) contains dentin canals. In the larva, these do not reach the enamel-dentin border but end below this border in front of a denser hard substance, possibly enameloid. The pedicel in the adult and the basal portion of the tooth in the larva (base) are without dentin canals. These parts of the teeth are characterized by longitudinally arranged collagen fibres as visualized on the demineralized surfaces. These observations indicate a congruency in early-larval and adult teeth between base and pedicel as well as apex and crown. This partition is also confirmed by the calcium and phosphate values which were identical in larvae and adults. Highest values are found in enamel and lowest values in the tooth-bearing bone. Calcium and phosphate content show a clear difference between dentin and the basal part of the tooth (pedicel and base). The ring-like dividing zone in the adult tooth is less well mineralized.