Resources and Information on Axolotls
This is catalogue of gathered articles, studies, and publishings that focus on everything relating to Ambystoma mexicanum. Axolotls have a very long, complex, and fascinating role in science, culturally, environmentally, and within the pet trade. These impactful animals have made a splash in various aspect of scientific research, primarily for their regenerative capabilities, but their unusual appearance has also captured the interest of millions of aquarium keepers worldwide for centuries!
Histories of Ambystoma mexicanum
"The Mexican axolotl (Ambystoma mexicanum) is an icon of culture, a revered aquarium pet, and a highly valued animal model in biomedical research. Unfortunately, Mexican axolotls are critically endangered in their natural Xochimilco habitat in Mexico City. If axolotls go extinct, current efforts to conserve the Xochimilico ecosystem will be undermined, as will efforts to genetically manage the laboratory populations that are needed to sustain research efforts around the world. A concerted global effort is needed to protect and manage this irreplaceable species in natural and laboratory environments."
"Ambystoma mexicanum (axolotl) embryos and juveniles have been used as model organisms for developmental and regenerative research for many years. This neotenic aquatic species maintains the unique capability to regenerate most, if not all, of its tissues well into adulthood. With large externally developing embryos, axolotls were one of the original model species for developmental biology... Here, we provide an updated axolotl-staging chart ranging from one-cell stage to immature adult, paired with a perspective on both historical and current axolotl research that spans from their use in early studies of development to the recent cutting-edge research, employment of transgenesis, high-resolution imaging, and study of mechanisms deployed in regeneration."
"Since 1245 up to the mid-twentieth century, when urban sprawl drove the axolotl to near-extinction, dwellers of the Basin of Mexico have eaten axolotls. (This Nahuatl word is pronounced ah-SHO-lotl). What this chapter explores is the possibility that by 1200 BC the Early Formative inhabitants of Lake Chalco also exploited the axolotl, probably for food. It suggests that the Formative Period dwellers of the Basin of Mexico devised a graphic symbol for the axolotl – in the form of its branchlike gill stalks. As the following study explains, the symbol may have referred to the axolotl as a food. It also may have signified the regenerative ability of this perpetually embryonic creature or the mistaken notion that the aquatic axolotl can transform into a terrestrial salamander. But before considering the early prehistory of the axolotl, the chapter looks at its “history” – what has been written about it since 1521 including its enormous bibliography in today’s scientific literature – and its role in Aztec mythology."
A transcribed entry by M. Auguste Duméril in 1866, which described the rudimentary experiments that he preformed, and observations of the captive axolotls that were imported from Mexico for the first time. His research sparked centuries of scientific research on regeneration and amphibians.
"Today the Mexican axolotl is critically endangered in its natural habitat in lakes around Mexico City, but thrives in research laboratories around the world, where it is used for research on development, regeneration, and evolution. Here, we concentrate on the early history of the axolotl as a laboratory animal to celebrate that the first living axolotls arrived in Paris in 1864, 150 years ago. Maybe surprisingly, at first the axolotl was distributed across Europe without being tied to specific research questions, and amateurs engaged in acclimatization and aquarium movements played an important role for the rapid proliferation of the axolotl across the continent. But the aquarium also became an important part of the newly established laboratory, where more and more biological and medical research now took place. Early scientific interest focused on the anatomical peculiarities of the axolotl, its rare metamorphosis, and whether it was a larva or an adult... Nowadays, technical developments such as transgenic lines, and the very strong interest in stem cell and regeneration research has again catapulted the axolotl into becoming an important laboratory animal."
"The salamander Ambystoma mexicanum, commonly called “the axolotl” has a long, illustrious history as a model organism, perhaps with one of the longest track records as a laboratory-bred vertebrate, yet it also holds a prominent place among the emerging model organisms. Or rather it is by now an “emerged” model organism, boasting a full cohort molecular genetic tools that allows an expanding community of researchers in the field to explore the remarkable traits of this animal including regeneration, at cellular and molecular precision—which had been a dream for researchers over the years. This chapter describes the journey to this status, that could be helpful for those developing their respective animal or plant models."
"One of many research questions investigated in the axolotl is regeneration. The species' astonishing ability to regenerate tissues and entire body parts already became apparent shortly after the first 34 living axolotls had been brought from Mexico to Europe in 1864. In the context of their unclear status as larvae or adults and the mysterious transformation of some animals into an adult form, the Paris zoologist Auguste Duméril cut off the gills of several individuals in an attempt to artificially induce the metamorphosis. This produced the first reports on the animals' regenerative powers and led to sporadic but continuous investigations. But it remained just one of the many phenomena studied in axolotls..."
A Closer Look at Axolotls
"The critical water quality parameters that directly affect the axolotl's health include water temperature, ammonia (NH3), nitrite (NO2-), nitrate (NO3-), pH, carbonate hardness (KH, also known as alkalinity), general hardness (GH, also known as permanent hardness) and dissolved oxygen (DO). Additionally, some parameters have diurnal fluctuations. Imbalances in any one of these parameters may have an impact on others and these inter-relationships will be explained. But before the specifics of each water quality parameter is discussed, they must understand the nitrogen cycle."
"The external gills of captive Mexican axolotl (Ambystoma mexicanum) sometimes develop atypical blood vessels, the cause of which is unknown. We observed the external gill filaments of an individual animal with dilated blood vessels that formed a semicircle within the filament tissue. The positioning of the swollen blood vessels compressed the adjacent capillaries and connective tissues. Normal external gill filaments in urodelans contain a blood-vessel system with afferent and efferent arterioles that connect to circumvent the outer gill periphery. We infer that the dilated blood vessels in the axolotl originated from these arterioles."
"The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging… In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth."
"Over a period of approximately 10 weeks from December 2009 until March 2010 a captive axolotl (Ambystoma mexicanum) was observed to change from a wild-type phenotype to a leucistic phenotype. It is common for leucistic axolotls to darken as they mature, but a change from wild-type to leucistic is a phenomenon that, to the author’s knowledge, has not been documented before. Over the winter of 2009 the axolotl began to lose pigmentation across its whole body becoming increasingly pale. Pigmentation loss began uniformly and the animal passed through stages of appearing brown and grey until it finally became fully white, with semi-translucent skin and pink gills. The dorsal surface retained small amounts of pigment giving a pale grey appearance.The pigment of the eyes remained unaffected, as found in leucistic morphs."
"To determine the protein nutritional requirements in juvenile axolotls (Ambystoma mexicanum) based on four isocaloric diets (8% lipids) with 30, 35, 30, and 45% protein: Six axolotls were used per test, during a period of 81 days. The diets were prepared using fishmeal as a protein source and fish oil as a lipid source. The feed was supplied every 48 h with 4% of the weight of the biomass of organisms per experimental reservoir. Four biometrics were performed throughout the experiment and growth parameters were determined: height, weight gained per day, specific growth rate, survival, Fulton’s K, and protein efficiency rate. The digestibility of each of the diets was also determined. Results: There were significant differences in the growth and survival of the axolotls, the diet with 45% protein showed the best growth results."
"Good nutritional husbandry is crucial to maintain high welfare standards in captive animals. Both direct effects of diet on growth, development, and maintenance, and indirect effects of feeding regimes on behavior may be important. Despite this, many questions remain as to how we should best feed many of the species that are commonly kept in captivity. There is a great deal of speculation amongst animal keepers as to issues such as whether a mixed diet is better than an invariant one, but little research is available to inform this question. In this study, we investigate the impact of mixed versus invariant diets on growth and behavior in the axolotl (Ambystoma mexicanum), an aquatic amphibian of severe conservation concern that is frequently maintained in captive collections. We then use our results to provide advice on feeding management in the context of improved welfare. We maintained juvenile axolotls under one of three ‘diets’ (feeding regimes): bloodworm (invariant), Daphnia (invariant), and alternating these two prey items between feeds (mixed). Morphological and behavioral data were collected over a period of 15 weeks and analyzed using generalized linear mixed models to determine whether our feeding treatments influenced growth and behavior. We find that axolotls grew fastest on our bloodworm diet and slowest on our Daphnia diet, with a mixed feeding regime leading to intermediate growth rates... Overall, our results suggest that mixed diets in themselves may not be beneficial to the growth or welfare of axolotls as compared to a high-quality invariant diet."
"Ambystoma mexicanum, a highly endangered species, is endemic to lake Xochimilco (Mexico City, Mexico) which currently is being negatively affected by the introduction of Oreochromis niloticus (Tilapia) and water pollution. During the first weeks of development, when mortality is the highest, Ambystoma mexicanum depends on a diet of zoo plankton. The aim of this study was to check whether contamination levels in lake Xochimilco influence zooplankton consumption by similar size classes of A. mexicanum and Oreochrois niloticus. In this study, we analysed changes in functional responses and prey preference of A. mexicanum and larval Tilapia in two media, one filtered lake Xochimilco water and another one with reconstituted water… Our functional response tests showed that regardless of the prey type, prey consumption by A. mexicanum was lower in contaminated water from lake Xochimilco… Our results indicate that both water quality of the lake and the presence of the more resistant exotic fish adversely impact the survival of this endangered amphibian. "
"For over a century, the axolotl egg, larva, and adult have been used to unravel the mysteries of development, metamorphosis, regeneration, and ontogenetic evolutionary transformations. Embryologically, the axolotl, like other amphibians, is an anamniote (lacking an amnion) that develops outside the maternal body from a spherical egg two millimeters in diameter..."
"Here, we tested the influence of size and ontogeny on suction feeding kinematics in adults, juveniles, and larvae of the axolotl (Ambystoma mexicanum) using high-speed video recordings. Our data show that size had an influence on nearly all kinematic variables examined, but kinematics often deviated from the predictions of simple geometric scaling models. Moreover, for both the velocity and acceleration of mouth and hyoid movements, the effect of size differed according to the developmental stage. While overall movements were faster in larger animals, the velocity of movement increased faster with size in adults. This could be explained by the fact that the skull undergoes changes at adulthood due to partial remodeling."
"The epidermal Leydig cells (LC) of larval and paedomorphic Urodela (= Caudata) are highly specialized cells, which are characterized by a complex peripheral cytoskeleton (Langerhans’ net) and numerous inclusions usually named secretory granules. We studied number, distribution and development of these cells in larvae up to 100 days after hatching and in some adults of the paedomorphic axolotl (Ambystoma mexicanum). With the exception of a short period after hatching, relation between age and total length of larvae was linear. The tail grew positively, the width of the head negatively allometric. Keeping larvae in groups resulted in a somewhat slower growth, in deviations from a strict linearity of some morphological parameters, and in a delayed increase of the number of LC, which is interpreted as crowding effect. LC could be identified already before hatching and developed first in the head, then in the trunk, and finally in the tail. Number of LC increased highly disproportionally during larval growth..."
"The present study aims to describe the morphological and histochemical changes in the epidermis of 10 cutaneous regions from juvenile (4 months old) and adult (24 and 48 months old) non-metamorphic A. mexicanum, with a particular focus on the amount and histochemical characteristics of LCs. Results indicate that the juvenile epidermis is a stratified cuboidal epithelium formed by three strata: basal, spinosum (containing the LCs), and apical. The most superficial layer contains cuboidal cells that lack the characteristics of a true stratum corneum. In adults, the stratum apical is also formed by squamous cells, suggesting a transition to a cornified and squamous layer as age increases...These natural axolotl epidermal changes indicate a gradual transition toward a morphology resembling metamorphic skin as age advances..."
"I questioned the salinity at which axolotl embryos
will experience decreased survivorship or increased morphological deformities. I used a freshwater solution as well as increasing saline solutions (with the highest being similar to the current salinity conditions in Laguna Alchichica), in order to determine at what point axolotl embryos cease to develop into viable young. I collected survival rates, developmental stages, number of deformities, and hatching rates as well as measurements of body length, head length, intraocular distance, and gill length of the hatchlings. The embryos reared in the 4 ppt NaCl/L solution showed the greatest amount of abnormalities, including many displaying a very distended, fluid filled abdominal cavity. Generally, embryos reared in the 1 ppt NaCl/L solution grew to be significantly larger than the others, based on the analysis of various two-tailed T-tests. The embryos reared in the saline concentration that was similar to that found in the lakes in central Mexico failed to develop."
"...There are three pigment cell types found in adult axolotl skin - melanophores, xanthophores and iridophores. Both pigments and pigment cells undergo specific developmental changes in axolotls. Melanophores are the predominant pigment cell type throughout development; xanthophores occur secondarily and in fewer numbers than melanophores; iridophores do not appear until well into the larval stage and remain thereafter as the least frequently encountered pigment cell type...This study forms the basis for comparison of the wild type pigment phenotype to the three mutant phenotypes-melanoid, axanthic and albino-found in the axolotl."
"Our survey recovered ca. 60% of Lake Xochimilco's historically recorded fish and amphibian species, including rare species and novel taxa not detected by past traditional surveys. However, our findings imply a severely degraded wetland, with alpha diversity indices indicating a low-diversity ecosystem dominated by alien fishes. Beta diversity analysis revealed a heterogeneous ecosystem that may be driven partially by the presence of alien fish, particularly cyprinids. Environmental variables linked to pollution predicted the presence of non-native fish families... Despite the ongoing degradation of this ecosystem, native and endemic fauna are persisting, although detections were typically rare. We found no evidence of the Critically Endangered axolotl salamanders (Ambystoma sp.) from wild sites; however, we detected their presence in one wildlife refuge, highlighting the potential of refuges to prevent complete extinction in the wild."
"This study evaluates the viability of restored and artificial wetlands for axolotl conservation by comparing movement patterns, home range sizes, and habitat use. Using VHF telemetry, we tracked captive-bred axolotls released into both environments. Axolotls survived and foraged successfully in both sites... Recaptured individuals gained weight, suggesting successful adaptation, although two axolotls were lost to avian predation in Xochimilco after the study concluded. These findings highlight the potential of artificial wetlands like La Cantera Oriente for axolotl conservation by providing stable conditions that may mitigate habitat degradation and climate change impacts. The study recommends integrating native and artificial habitats into conservation strategies, incorporating predator awareness training before release, and ongoing habitat monitoring to enhance survival outcomes for this iconic species."
"... In addition, we have recently shown that a population of UV photoreceptors is also present in the retina of the neotenic Mexican salamander Ambystoma mexicanum. In this article we have presented a summary of our experiments as well a discussion of their implications for research concerning the visual system of urodeles. We have attempted to present a simplified version of our techniques so that those unfamiliar with visual physiology can gain an understanding of the experiments."
"The Axolotl, Ambystoma mexicanum, endemic to the freshwater lakes, Xochimilco and Chalco in Mexico City, feeds on zooplankton during its larval stages. We evaluated the functional response over eight weeks of A. mexicanum fed different prey items found in its natural habitat (rotifers: Brachionus havanaensis, B. calyciflorus, B. rubens and Plationus patulus; cladocerans: Moina macrocopa, Macrothrix triserialis, Alona glabra and Simocephalus vetulus; and ostracods: Heterocypris incongruens). Zooplankton consumption by A. mexicanum varied in relation to the prey species and age of the larvae. Unlike oviparous fish larvae which often feed preferentially on rotifers in the first few weeks, A. mexicanum larvae fed more on cladocerans and ostracods. Among the cladocerans offered, larval A. mexicanum consumed higher numbers of M. triserialis and M. macrocopa. Feeding on the largest cladoceran tested, S. vetulus, increased after the fifth week. There was a consistent increase in the number of ostracods Heterocypris incongruens, consumed with age, from 4 to 169 prey per larva over eight weeks. The results are discussed with relation to the importance of zooplankton diet in conservation effort of this endangered species in Lake Xochimilco."
"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."
"The Mexican axolotl, Ambystoma mexicanum is an aquatic-phase oxygen conformer with a close correlation between rates of aquatic oxygen consumption and aquatic oxygen tension. Rates of oxygen consumption at normoxic oxygen tensions were 1 8.2μ1 02.g·1.h-1 at 20°C and 1 3.8μ.I 02.g-1.h-1 at 30°C. Air-breathing (i.e. rising to the water surface to gulp air) increases in hypoxia, associated with a decrease in gill ventilatory movements. Therefore, oxygen compensation during aquatic hypoxia appears to occur in the aerial phase, whilst aquatic gas-exchange surfaces show little ability to compensate for changes in ambient oxygen tension."
"Temperature can affect biological processes in the body such as metabolism, growth and reproduction. Just like other Caudata orders, axolotls are very sensitive to temperature changes. Water temperature greatly affects the growth and development of axolotls. Axolotls are cold-blooded animals that cannot adjust their body temperature to the temperature of the water where they live. Axolotls are very dependent on water temperature, if axolotls live at low water temperatures, their metabolism will decrease and they cannot digest food perfectly. Conversely, if they live at higher temperatures, axolotls will increase their appetite and cause a wasteful energy burning process. High temperatures can cause axolotls to become stressed quickly and shorten their lifespan."
"In this work we analyze axolotl embryo development, identifying and describing the stages from zygote until hatching with high quality images. The embryos were maintained in natural conditions at 16°C, inside their jelly layers the whole study period to take images that show the natural development from oviposition to hatching We analyzed and described a total of 49 development stages that were grouped in 5 phases: (1) fertilized oocyte, (2) cleavage, (3) gastrulation, (4) neurulation, and (5) organogenesis. Time from fertilization to hatching took about 350 hours under the management conditions set up in laboratory; it can be considered that the complete development of the axolotl embryo until hatching takes two weeks approximately."
"The epidermis of the adult axolotl, like that of larval salamanders, contains numerous, large, club-like Leydig cells which resemble the unicellular glands found in fishes (Parakkal & Alexander, 1972). These cells have been studied in different larva land adult urodeles (Leydig, 1857; Langerhans, 1873; Pfitzner, 1879; Dawson, 1920;Hay, 1961; Kelly, 1966). Since their secretion has not been seen escaping onto the skin surface, it has been presumed to be released within the epidermis to provide protection from bacteria and viruses (Andrew & Hickman, 1974).The objective of the present study was to elucidate the fine structure of the Leydig cells in the epidermis of neotenous adult axolotls using more effective, modern fixatives and to readdress the question of whether their secretory product is released within the epidermis as a fluid reserve or is discharged onto the surface of the skin."
"The lateral line system of axolotls, like that of most other salamanders, anuran larvae, and fishes, consists of numerous epidermal sensory organs arranged in a well-defined lines that extend over the whole body surface. The distribution and development of these sensory organs in salamanders have been studied extensively and have contributed much to our understanding of the structure and function of lateral line systems in anamniotes... Details of the histology, distribution, and innervation of the lateral line organs of axolotls will be summarized..."
"These mechanoreceptive organs comprise a centrally elongated strip of directionally sensitive hair cells surrounded by a peripheral zone of support cells and are stimulated by low frequency water movements parallel to the major axes of the receptors. Pit organs are a second class of smaller mechanoreceptors, with fewer hair cells that have lower displacement thresholds to low frequency wave stimuli. In salamanders and in most nonteleost fishes, an additional class of lateral line receptors, termed ampullary organs, occur on the head adjacent to the cephalic lines of neuromasts and pit organs. Ampullary organs are electroreceptors, sensitive to the low frequency DC fields generated by other living organisms and are believed to detect potential prey."
"The present study investigates when the two populations of neural crest derived chromatophores, melanophores and xanthophores become determined and how 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... 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."
Focusing on Regeneration
"Multiple factors are thought to cause limb abnormalities in amphibian populations by altering processes of limb development and regeneration. We examined adult and juvenile axolotls (Ambystoma mexicanum) in the Ambystoma Genetic Stock Center (AGSC) for limb and digit abnormalities to investigate the probability of normal regeneration after bite injury. We observed that 80% of larval salamanders show evidence of bite injury at the time of transition from group housing to solitary housing. Among 717 adult axolotls that were surveyed, which included solitary-housed males and group-housed females, approximately half presented abnormalities, including examples of extra or missing digits and limbs, fused digits, and digits growing from atypical anatomical positions. Bite injury probably explains these limb defects, and not abnormal development, because limbs with normal anatomy regenerated after performing rostral amputations. We infer that only 43% of AGSC larvae will present four anatomically normal looking adult limbs after incurring a bite injury. Our results show regeneration of normal limb anatomy to be less than perfect after bite injury."
"Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye..."
"Mexican axolotls lose potential for lens regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post-hatching) and old (3 months post-hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with lens regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions."
"In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body..."
"Interestingly, axolotls never seem to form scar tissue at the site of amputation once regeneration is completed. Before now, very few studies were directly focused on the description of the events happening during wound healing after a skin injury in salamanders. In this paper, we directly investigated skin wound healing after excisional wounding which removed the epidermis, dermis and basement membrane in the axolotl. Axolotls were wounded with a 1.5-mm skin biopsy punch. Results show rapid re-epithelialization of the wound within 8 hrs after wounding. Histological analysis of wound healing confirmed the absence of tissue fibrosis throughout the process and shows that skin integrity is re-established by 90 days after wounding. Results also reveal the absence of neutrophils in the wound area, suggestive of a lack of or low inflammatory response..."
"To date, studies investigating axolotl spinal cord regeneration have placed particular emphasis on understanding how cells immediately adjacent to the injury site respond to damage to promote regenerative repair. How neurons outside of this immediate injury site respond to an injury remains unknown. Here, we identify a population of dpErk+/etv1+ glutamatergic neurons in the axolotl telencephalon that are activated in response to injury and are essential for tail regeneration. Furthermore, these neurons project to the hypothalamus where they upregulate the neuropeptide neurotensin in response to injury. Together, these findings identify a unique population of neurons in the axolotl brain whose activation is necessary for successful tail regeneration, and sheds light on how neurons outside of the immediate injury site respond to an injury."
"The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers."
"The axolotl Ambystoma mexicanum is one of the most commonly used model organisms in developmental and regenerative studies because it can reconstitute what is believed to be a completely normal anatomical and functional forelimb/hindlimb after amputation. However, to date it has not been confirmed whether each regenerated forelimb muscle is really a “perfect” copy of the original muscle. This study describes the regeneration of the arm, forearm, hand, and some pectoral muscles (e.g., coracoradialis) in transgenic axolotls that express green fluorescent protein (GFP) in muscle fibers. The observations found that: there were muscle anomalies in 43% of the regenerated forelimbs..."
"The axolotl Ambystoma mexicanum is one of the most used model organisms in developmental and regenerative studies because it is commonly said that it can reconstitute a normal and fully functional forelimb/hindlimb after amputation. However, there is not a publication that has described in detail the regeneration of the axolotl hindlimb muscles. Here we describe and illustrate, for the first time, the regeneration of the thigh, leg and foot muscles in transgenic axolotls that express green fluorescent protein in muscle fibers and compare our results with data obtained by us and by other authors about axolotl forelimb regeneration and about fore- and hindlimb ontogeny in axolotls, frogs and other tetrapods."
"Here, we probed the limits of axolotl limb regeneration by challenging them with repeated amputation. We observed both a decline in regenerative fidelity as well as ability to regenerate beyond the plane of amputation. These findings suggest that there is indeed a limit to the ability of axolotl limbs to continue to regenerate with near perfection. Furthermore, our studies with repeated, serially distal amputations suggest that the decline may be due in part to recurrently injuring the same tissue instead of globally exhausting the regenerative cycle. One longstanding hypothesis in the field of regenerative biology is that there is a “tug of war” between the scarring process and regenerative process, and humans may possess limited capabilities because the molecular programs that drive the former process win this competition following injury. In support of this, we found evidence of extensive collagen deposition in limbs that failed to regenerate after repeated amputation."
"Here, we discovered a role for the sympathetic nervous system in stimulating a body-wide stem cell activation response to amputation that drives enhanced limb regeneration in axolotls. This response is mediated by adrenergic signaling, which coordinates distant cellular activation responses via the α2Α-adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α2A- and β-adrenergic signaling act upstream of mTOR signaling. Notably, systemically activated axolotls regenerate limbs faster than naïve animals, suggesting a potential selective advantage in environments where injury from cannibalism or predation is common. This work challenges the predominant view that cellular responses underlying regeneration are confined to the injury site and argues instead for body-wide cellular priming as a foundational step that enables localized tissue regrowth."
"The mechanisms underlying scarless versus fibrotic wound healing remain a critical challenge in regenerative medicine. To elucidate the mechanisms of scarless repair, the axolotl (Ambystoma mexicanum), a model organism with exceptional regenerative capacity, has gained increasing prominence. Although axolotls are capable of regenerating complex structures such as limbs and tails, whether their skin regeneration is uniformly scarless—especially across different anatomical sites—remains undefined. Here, we demonstrate that adult axolotl tail skin achieves scarless regeneration, while dorsal skin repair results in fibrotic scarring..."
"Connective tissues—skeleton, dermis, pericytes, fascia—are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation..."
Genetic Information
"Several dozen Mendelian mutants have been discovered in axolotl (Ambystoma mexicanum) populations, including several that affect pigmentation. Four recessive mutants have been described in the scientific literature and genes for three of these have been identified. Here we describe and genetically dissect copper, a mutant with an albino-like phenotype known only from the pet trade. We performed a cross segregating copper and wildtype color phenotypes and used bulked segregant RNA-Seq to identify a region on chromosome 6 that was enriched for single-nucleotide polymorphisms (SNPs) between the color phenotypes. This region included Tyrosinase-like Protein 1 (Tyrp1), a melanin synthesis protein that when mutated, is associated with lighter than black melanin coloration in animal models and oculocutaneous albinism in humans. Inspection of RNA-Seq reads identified a single nucleotide deletion that is predicted to change the coding frame, introduce a premature stop codon in exon 6 and yield a truncated Tyrp1 protein in copper individuals. Using CRISPR-Cas9 editing, we show that wildtype Tyrp1 crispants exhibit copper pigmentation, thus confirming Tyrp1 as the copper locus. Our results suggest that commercial and hobbyist axolotl populations may harbor useful mutants for biological research."
"Axolotls with abnormal limb development and renal failure were noted in Rufus R. Humphrey's axolotl colony as early as 1949 (at the University of Rochester). The source of short toes animals was probably white animals obtained by Humphrey from the Effingham B. Morris Farm of the Wistar Institute (in Bristol, Pennsylvania, USA)… Humphrey obtained the female progenitor in 1935. By 1958 it was concluded that the abnormalities had a genetic basis. Humphrey published a description and experimental analysis of short toes in 1967 at Indiana University (Humphrey 1967). Breeding showed the trait to be homozygous recessive in leucistic (white, d) genetic background animals affecting limbs and the urogenital system, becoming lethal due to eventual renal failure. He concluded that it was not linked to the white (d) locus."
"The development of transgenesis in axolotls is crucial for studying development and regeneration as it would allow for long-term cell fate tracing as well as gene expression analysis. We demonstrate here that plasmid injection into the one-cell stage axolotl embryo generates mosaic transgenic animals that display germline transmission of the transgene... This represents the first demonstration in the axolotl of germline transmission of a transgene. Using this technique we have generated a germline transgenic animal expressing GFP ubiquitously in all tissues examined. We have used this animal to study cell fate in the dorsal fin during development. We have uncovered a contribution of somite cells to dorsal fin mesenchyme in the axolotl, which was previously assumed to derive solely from neural crest. We have also studied the role of blood during tail regeneration by transplanting the ventral blood-forming region from GFP+ embryos into unlabeled hosts. During tail regeneration, we do not observe GFP+ cells contributing to muscle or nerve, suggesting that during tail regeneration blood stem cells do not undergo significant plasticity."
"Injection of RNA, DNA, or morpholinos into amphibian and fish embryos is a standard technique used to test gene function during development or to make transgenic animals. The techniques used to inject axolotl (Ambystoma mexicanum) embryos are very similar to Xenopus embryos... Injection of plasmid DNA into the axolotl egg results in varying frequencies of integration (depending on the plasmid DNA) within one of the first few cell divisions."
"Urodele amphibians are unique among adult vertebrates in their ability to regenerate missing limbs. The process of limb regeneration requires several key tissues including a regeneration-competent wound epidermis called the regeneration epithelium (RE). We used microarray analysis to profile gene expression of the RE in the axolotl, a Mexican salamander. A list of 125 genes and expressed sequence tags (ESTs) showed a >1.5-fold expression in the RE than in a wound epidermis covering a lateral cuff wound. A subset of the RE ESTs and genes were further characterized for expression level changes over the time-course of regeneration. This study provides the first large scale identification of specific gene expression in the RE."
"The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning–candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl."
"The great diversity of color patterns observed among amphibians is largely explained by the differentiation of relatively few pigment cell types during development. Mexican axolotls present a variety of color phenotypes that span the continuum from leucistic to highly melanistic. The melanoid axolotl is a Mendelian variant characterized by large numbers of melanophores, proportionally fewer xanthophores, and no iridophores. Early studies of melanoid were influential in developing the single-origin hypothesis of pigment cell development, wherein it has been proposed that all three pigment cell types derive from a common progenitor cell, with pigment metabolites playing potential roles in directing the development of organelles that define different pigment cell types. Specifically, these studies identified xanthine dehydrogenase (XDH) activity as a mechanism for the permissive differentiation of melanophores at the expense of xanthophores and iridophores. We used bulked segregant RNA-Seq to screen the axolotl genome for melanoid candidate genes and identify the associated locus. Dissimilar frequencies of single-nucleotide polymorphisms were identified between pooled RNA samples of wild-type and melanoid siblings for a region on chromosome 14q. This region contains gephyrin (Gphn), an enzyme that catalyzes the synthesis of the molybdenum cofactor that is required for XDH activity, and leukocyte tyrosine kinase (Ltk), a cell surface signaling receptor that is required for iridophore differentiation in zebrafish. Wild-type Ltk crispants present similar pigment phenotypes to melanoid, strongly implicating Ltk as the melanoid locus. In concert with recent findings in zebrafish, our results support the idea of direct fate specification of pigment cells and, more generally, the single-origin hypothesis of pigment cell development."
"The ability to generate transgenic animals sparked a wave of research committed to implementing such technology in a wide variety of model organisms. Building a solid base of ubiquitous and tissue-specific reporter lines has set the stage for later interrogations of individual cells or genetic elements. Compared to other widely used model organisms such as mice, zebrafish and fruit flies, there are only a few transgenic lines available in the laboratory axolotl (Ambystoma mexicanum), although their number is steadily expanding. In this review, we discuss a brief history of the transgenic methodologies in axolotl and their advantages and disadvantages. Next, we discuss available transgenic lines and insights we have been able to glean from them. Finally, we list challenges when developing transgenic axolotl, and where further work is needed in order to improve their standing as both a developmental and regenerative model."
"We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible."
"Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged
Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5–15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration."
Infectious Diseases and Disorders
"Ambystoma mexicanum kept as pets are affected by a variety of diseases. However, no reports regarding the incidence of specific diseases are available. This study aimed to identify the diseases that occur frequently in this species by surveying the incidence of conditions in pet A. mexicanum specimens brought to a veterinary hospital. The sample comprised 97 pet A. mexicanum individuals brought to the authors’ hospital during the 82-month period, i.e., from January 2008 to October 2014. In total, 116 diseases were identified. The most common disease was hydrocoelom (32 cases; 27.5% of all cases). Elucidating the pathogenesis of hydrocoelom, which has a high prevalence rate, is vital to maintaining the long-term health of A. mexicanum pets."
"The Ambystoma mexicanum axolotl is a highly threatened amphibian and a valuable research model, with very little information about bacterial diseases affecting it. The aim of this study was to perform an identification of bacteria responsible for septicemia in three individuals. For all of them, necropsies were made, bacteria classification was performed by traditional and DNA-based molecular methods and tissues were histologically examined. All animals showed edema and ascites, and other tissues such as the lungs, spleen, liver, and kidney were also affected, dermatitis also occurred, in one case, the dermatitis was severe."
"A 3-year-old axolotl (Ambystoma mexicanum) presented with anorexia, weakness and abdominal swelling due to hydrocoelom. According to the anamnesis, the symptoms had occurred 3 months prior. The radiographic and ultrasonographic examinations revealed that these symptoms resulted from the fluid accumulating in the coelomic cavity. Then, the habitat of the axolotl and the fluid in the coelomic cavity were inspected. Then, the patient's hydrocoelomic fluid was drained and medical treatment was performed. In addition, habitat arrangement and diet were changed. At the follow-up visit 1 week later, the patient reported a significant decrease in hydrocoelom and a significant increase in appetite and mobility. Laboratory results showed no microbiologic growth in the sample taken from the coelomic cavity... In conclusion, non-infective hydrocoelom was observed in an axolotl, and medical treatment was performed."
"From October 2001 through February 2002, we had a larger than normal number of axolotls and were forced to house some animals in unusually crowded conditions. During this period a disease outbreak affecting the entire colony occurred. Juvenile and adult axolotls showed skin pustules, reddening and swelling of the vent, limbs and neck, and occasional bleeding from the gills. Any animals that exhibited these signs died within 1-5 days. We had experienced a similar disease in the summer of 1995, following the introduction in late 1994 of wild axolotls from Lake Xochilmilco near Mexico City. The colony was experiencing similar high-density conditions and we were also housing tiger salamanders. The disease moved through the entire colony in a 4-5 month period and then the incidence tapered off. Careful husbandry and immediate removal of animals showing clinical signs eventually extinguished the disease"
Amphibians and Other Ambystoma Species
"In this overview, we briefly review certain features of mammalian immune systems (covered in much more detail in virtually any textbook of immunology), summarize the evidence that salamanders have a subdued immune system (based on functional evidence mostly from the works of Delanney, Cohen, Charlemagne, and Tournefier), describe the unpublished data that we and our collaborators have generated to show that the axolotl has both a Major Histocompatibility Complex (MHC) and functional T lymphocytes, and conclude with our present interpretation of why the axolotl and other urodeles have subdued immune responses."
"...Caudates (salamanders) in the genus Ambystoma represent a challenge for rearing because of their aggressive nature, and several recent projects have found higher than normal stress levels in captive-reared salamanders. In this study I compared various captive-rearing scenarios designed to minimize stress levels in Spotted Salamanders (A. maculatum). I reared larvae in five treatments, three that varied in starting density (six, 12, and 30 larvae per 1000 L tank), one with high food, and one with added environmental complexity to the tanks (sticks and refugia). After metamorphosis, I weighed the salamanders and collected blood samples to determine the relative abundance of two white blood cell types (neutrophils and lymphocytes) to calculate neutrophil-lymphocyte (N-L) ratios, which covary with stress hormone levels… The treatment that led to the largest salamanders overall and the only one where N-L ratios were similar to those from the wild was the treatment with the lowest starting density (six larvae per 1000 L tank). Furthermore, N-L ratios across all treatments were a direct function of body size; larger salamanders exhibited lower stress."
This catalogue defines the 29 species of Ambystoma salamanders found in the Americas around 1969. Physical descriptions, habitat distribution, fossil records, etymology, and relevant history are discussed.
"Amphibians are sensitive to contaminants, including NaCl, because of their porous skin and osmoregulatory adaptations to freshwater. However, similarities and differences between effects of wastewaters and road salts have not been investigated. Therefore, we investigated the relative influence of wastewaters and NaCl at equivalent concentrations of chloride on three larval amphibian species that occur in areas with increased salinity. We determined acute toxicity and growth effects on Boreal Chorus Frogs (Pseudacris maculata), Northern Leopard Frogs (Rana pipiens), and Barred Tiger Salamanders (Ambystoma mavortium). We posited that wastewaters would have additive effects on amphibians compared to NaCl because wastewaters often have additional toxic heavy metals and other contaminants. For NaCl, toxicity was higher for frogs than the salamander... The negative influence of salts on imperiled amphibians underscores the need to restore landscapes with increased salinity and reduce future salinization of freshwater ecosystems."