The metamorphosis of amphibian myocardium: moving to the heart of the matter.

Amphibians are a classical object for physiological studies, and they are of great value for developmental studies owing to their transition from an aquatic larval form to an adult form with a terrestrial lifestyle. Axolotls (Ambystoma mexicanum) are of special interest for such studies because of their neoteny and facultative pedomorphosis, as in these animals, metamorphosis can be induced and fully controlled in laboratory conditions. It has been suggested that their metamorphosis, associated with gross anatomical changes in the heart, also involves physiological and electrical remodeling of the myocardium. We used whole-cell patch clamp to investigate possible changes caused by metamorphosis in electrical activity and major ionic currents in cardiomyocytes isolated from paedomorphic and metamorphic axolotls. T4-induced metamorphosis caused shortening of atrial and ventricular action potentials (APs), with no changes in resting membrane potential or maximum velocity of AP upstroke, favoring higher heart rate possible in metamorphic animals. Potential-dependent potassium currents in axolotl myocardium were represented by delayed rectifier currents IKr and IKs, and upregulation of IKs caused by metamorphosis probably underlies AP shortening. Metamorphosis was associated with downregulation of inward rectifier current IK1, probably serving to increase the excitability of myocardium in metamorphic animals. Metamorphosis also led to a slight increase in fast sodium current INa with no changes in its steady-state kinetics and to a significant upregulation of ICa in both atrial and ventricular cells, indicating stronger Ca2+ influx for higher cardiac contractility in metamorphic salamanders. Taken together, these changes serve to increase cardiac reserve in metamorphic animals.

The amazing and anomalous axolotls as scientific models.

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. However, increased access to, and use of, organisms with sequenced and annotated genomes, such as Xenopus laevis and tropicalis and Danio rerio, reduced the prevalence of axolotls as models in embryogenesis studies. Recent sequencing of the large axolotl genome opens up new possibilities for defining the recipes that drive the formation and regeneration of tissues like the limbs and spinal cord. However, to decode the large A. mexicanum genome will take a herculean effort, community resources, and the development of novel techniques. 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.

The use of transgenics in the laboratory axolotl.

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.

Axolotl: A resourceful vertebrate model for regeneration and beyond.

The regenerative capacity varies significantly among the animal kingdom. Successful regeneration program in some animals results in the functional restoration of tissues and lost structures. Among the highly regenerative animals, axolotl provides multiple experimental advantages with its many extraordinary characteristics. It has been positioned as a regeneration model organism due to its exceptional renewal capacity, including the internal organs, central nervous system, and appendages, in a scar-free manner. In addition to this unique regeneration ability, the observed low cancer incidence, its resistance to carcinogens, and the reversing effect of its cell extract on neoplasms strongly suggest its usability in cancer research. Axolotl's longevity and efficient utilization of several anti-aging mechanisms underline its potential to be employed in aging studies.

Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

DNA repair during regeneration in Ambystoma mexicanum.

The remarkable regenerative capabilities of the salamander Ambystoma mexicanum have turned it into one of the principal models to study limb regeneration. During this process, a mass of low differentiated and highly proliferative cells, called blastema, propagates to reestablish the lost tissue in an accelerated way. Such a process implies the replication of a huge genome, 10 times larger than humans, with about 65.6% of repetitive sequences. These features make the axolotl genome inherently difficult to replicate and prone to bear mutations. In this context, the role of DNA repair mechanisms acquires great relevance to maintain genomic stability, especially if we consider the necessity of ensuring the correct replication and integrity of such a large genome in the blastema cells, which are key for tissue regeneration. On the contrary, DNA damage accumulation in these cells may result in senescence, apoptosis and premature differentiation, all of them are mechanisms employed to avoid DNA damage perpetuation but with the potential to affect the limb regeneration process. Here we review and discuss the current knowledge on the implications of DNA damage responses during salamander regeneration.

Essential roles of matrix metalloproteinases in axolotl digit regeneration.

Among vertebrates, urodele amphibians possess a unique ability to regenerate various body parts including limbs. However, reports of their digit regeneration remain scarce, especially information about the related genes. In this study, it was evident that matrix metalloproteinases (mmps) including mmp9, mmp3/10a, and mmp3/10b, which play a crucial role in tissue remodeling, are highly expressed during early stages of digit regeneration in axolotl. Using in situ hybridization, we revealed that wound epidermis and blastema are two major origins of the MMPs during the regeneration process. Additionally, we found that the inhibition of MMPs with GM6001 (a wide-spectrum inhibitor of MMPs) in vivo after amputation disturbed normal digit regeneration process and resulted in malformed regenerates. Furthermore, inhibition of MMPs hindered blastema formation and decreased cell apoptosis at early stages in the digit regenerates. All these points suggest that MMPs are required for digit regeneration, as they play a significant role in the regulation of blastema formation.

Biology of primordial germ cells in vertebrates with emphasis in urodeles amphibians.

Primordial germ cells (PGCs) are highly specialized cells that play a relevant role in the maintenance and evolution of the species, since they create new combinations of genetic information between the organisms. Amphibians are a class of amniote vertebrates that are divided into three subclasses, the anurans (frogs and toads), the urodeles (salamanders and newts), and the gymnophiones (caecilians). The study of PGCs in amphibians has been addressed in more detail in anurans while little is known about the biology of this cell lineage in urodeles. Studies in some urodeles species have suggested that PGCs are of mesodermal origin, specifying in the lateral plate mesoderm at the late gastrula stage. With classical experiments it shown that, there is an induction of mesoderm, therefore most likely urodeles PGCs develop from unspecialized mesodermal tissue that responds to extracellular signals. However, some fundamental biological processes of PGCs such as the analysis of their specification, arrival, and colonization to the gonads, and their maintenance and differentiation into mature and fertile gametes remain to be elucidated. Therefore, knowledge about the biology of PGCs is of great importance to ensure the perpetuation of urodeles amphibians, as some species are in danger of becoming extinct.

Normal development in Ambystoma mexicanum: A complementary staging table for the skull based on Alizarin red S staining.

BACKGROUND: As the role of Ambystoma mexicanum, or the Mexican axolotl, expands in research applications beyond its traditional use in studies of limb regeneration, a staging table that is more anatomically extensive is required. Here, we describe axolotl skull development as it relates to previously established developmental stages that were based on limb development. RESULTS: We find that most key developmental events in the skull correspond to these previously established stages, creating easily recognizable stages of axolotl throughout skull morphogenesis. CONCLUSIONS: With this complementary staging table in hand, researchers can stage axolotl larvae when limb data are missing or incomplete, or when cranial data alone is available.

Axolotl Ambystoma mexicanum extract induces cell cycle arrest and differentiation in human acute myeloid leukemia HL-60 cells.

Acute myeloid leukemia is the most common form of acute leukemia in adults, constituting about 80% of cases. Although remarkable progress has been made in the therapeutic scenario for patients with acute myeloid leukemia, research and development of new and effective anticancer agents to improve patient outcome and minimize toxicity is needed. In this study, the antitumor activity of axolotl (AXO) Ambystoma mexicanum crude extract was assessed in vitro on the human acute myeloid leukemia HL-60 cell line. The anticancer activity was evaluated in terms of ability to influence proliferative activity, cell viability, cell cycle arrest, and differentiation. Moreover, gene expression analysis was performed to evaluate the genes involved in the regulation of these processes. The AXO crude extract exhibited antiproliferative but not cytotoxic activities on HL-60 cells, with cell cycle arrest in the G0/G1 phase. Furthermore, the AXO-treated HL-60 cells showed an increase in both the percentage of nitroblue tetrazolium positive cells and the expression of CD11b, whereas the proportion of CD14-positive cells did not change, suggesting that extract is able to induce differentiation toward the granulocytic lineage. Finally, the treatment with AXO extract caused upregulation of CEBPA, CEBPB, CEBPE, SPI1, CDKN1A, and CDKN2C, and downregulation of c-MYC. Our data clearly show the potential anticancer activity of Ambystoma mexicanum on HL-60 cells and suggest that it could help develop promising therapeutic agents for the treatment of acute myeloid leukemia.

Amputation-induced reactive oxygen species signaling is required for axolotl tail regeneration.

BACKGROUND: Among vertebrates, salamanders are unparalleled in their ability to regenerate appendages throughput life. However, little is known about early signals that initiate regeneration in salamanders. RESULTS: Ambystoma mexicanum embryos were administered tail amputations to investigate the timing of reactive oxygen species (ROS) production and the requirement of ROS for regeneration. ROS detected by dihydroethidium increased within minutes of axolotl tail amputation and levels remained high for 24 hr. Pharmacological inhibition of ROS producing enzymes with diphenyleneiodonium chloride (DPI) and VAS2870 reduced ROS levels. Furthermore, DPI treatment reduced cellular proliferation and inhibited tail outgrowth. CONCLUSIONS: The results show that ROS levels increase in response to injury and are required for tail regeneration. These findings suggest that ROS provide instructive, if not initiating cues, for salamander tail regeneration. Developmental Dynamics 248:189-196, 2019. © 2018 Wiley Periodicals, Inc.

Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration.

Epimorphic regeneration proceeds with or without formation of a blastema, as observed for the limb and skin, respectively. Inhibition of epimorphic regeneration provides a means to interrogate the cellular and molecular mechanisms that regulate it. In this study, we show that exposing amputated limbs to beryllium nitrate disrupts blastema formation and causes severe patterning defects in limb regeneration. In contrast, exposing full-thickness skin wounds to beryllium only causes a delay in skin regeneration. By transplanting full-thickness skin from ubiquitous GFP-expressing axolotls to wild-type hosts, we demonstrate that beryllium inhibits fibroblast migration during limb and skin regeneration in vivo Moreover, we show that beryllium also inhibits cell migration in vitro using axolotl and human fibroblasts. Interestingly, beryllium did not act as an immunostimulatory agent as it does in Anurans and mammals, nor did it affect keratinocyte migration, proliferation or re-epithelialization, suggesting that the effect of beryllium is cell type-specific. While we did not detect an increase in cell death during regeneration in response to beryllium, it did disrupt cell proliferation in mesenchymal cells. Taken together, our data show that normal blastema organogenesis cannot occur without timely infiltration of local fibroblasts and highlights the importance of positional information to instruct pattern formation during regeneration. In contrast, non-blastemal-based skin regeneration can occur despite early inhibition of fibroblast migration and cell proliferation.

Activation of Smad2 but not Smad3 is required to mediate TGF-ß signaling during axolotl limb regeneration.

Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-ß1 is highly upregulated during regeneration and that TGF-ß signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-ß antagonist SB-431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-ß during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-ß targets (e.g. Mmp13) are unaffected. This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-ß signaling supporting the regenerative process.

ENDOSCOPIC REMOVAL OF A FOREIGN BODY IN A MEXICAN AXOLOTL (AMBYSTOMA MEXICANUM) WITH THE USE OF MS222-INDUCED IMMOBILIZATION.

This communication briefly describes the use of tricaine methanesulfonate (MS222) to induce chemical restraint/general anesthesia of a Mexican axolotl (Ambystoma mexicanum) for the endoscopic retrieval of a gastric foreign body. There is very little published scientific literature concerning the anesthesia of Mexican axolotls. The anesthesia used in this case was an immersion bath of tricaine methanesulfonate where the concentration of tricaine methanesulfonate was gradually increased to 500 mg/L (ppm) over a 15-min period. A loss of righting reflex was observed within 3 min of attaining the final concentration of the anesthetic bath. The first voluntary movements following the transfer to a freshwater bath occurred within 7 min. The recovery was uneventful. Tricaine methanesulfonate in this case proved to be an effective anesthetic agent for a short, minimally invasive procedure.

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

BACKGROUND: AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. RESULTS: In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. CONCLUSIONS: Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

Buoyancy disorders in pet axolotls Ambystoma mexicanum: three cases.

As far as we are aware, there are no previous reports on the pathologic conditions of buoyancy disorders in Ambystoma mexicanum. Herein, we describe various clinical test results, clinical outcomes, and the pathological findings of an experimental pneumonectomy procedure in 3 A. mexicanum exhibiting abnormal buoyancy. The 3 pet A. mexicanum were adults, and their respective ages and body weights were 1, 5, and 6 yr and 48, 55, and 56 g. Two of these cases were confirmed via radiographic examination to have free air within the body cavity, and all 3 cases were found via ultrasonography to have an acoustic shadow within the body cavity and were diagnosed with pneumocoelom. Lung perforations were detected macroscopically in 2 of the cases, and all 3 cases had fibrosis in the caudal ends of the lungs. Removal of the lung lesions eliminated the abnormal buoyancy in all 3 cases. We concluded that air had leaked into the body cavity from the lungs, and we propose that lung lesions are an important cause of buoyancy disorders in A. mexicanum.

Analysis of signal processing in vestibular circuits with a novel light-emitting diodes-based fluorescence microscope.

Optical visualization of neural network activity is limited by imaging system-dependent technical tradeoffs. To overcome these constraints, we have developed a powerful low-cost and flexible imaging system with high spectral variability and unique spatio-temporal precision for simultaneous optical recording and manipulation of neural activity of large cell groups. The system comprises eight high-power light-emitting diodes, a camera with a large metal-oxide-semiconductor sensor and a high numerical aperture water-dipping objective. It allows fast and precise control of excitation and simultaneous low noise imaging at high resolution. Adjustable apertures generated two independent areas of variable size and position for simultaneous optical activation and image capture. The experimental applicability of this system was explored in semi-isolated preparations of larval axolotl (Ambystoma mexicanum) with intact inner ear organs and central nervous circuits. Cyclic galvanic stimulation of semicircular canals together with glutamate- and γ-aminobutyric acid (GABA)-uncaging caused a corresponding modulation of Ca(2+) transients in central vestibular neurons. These experiments revealed specific cellular properties as well as synaptic interactions between excitatory and inhibitory inputs, responsible for spatio-temporal-specific sensory signal processing. Location-specific GABA-uncaging revealed a potent inhibitory shunt of vestibular nerve afferent input in the predominating population of tonic vestibular neurons, indicating a considerable impact of local and commissural inhibitory circuits on the processing of head/body motion-related signals. The discovery of these previously unknown properties of vestibular computations demonstrates the merits of our novel microscope system for experimental applications in the field of neurobiology.

The amazing axolotl: robust kidney regeneration following acute kidney injury.

The incidence of kidney disease from acute and chronic conditions continues to escalate worldwide. Interventions to replace renal function after organ failure remain limited to dialysis or transplantation, as human kidneys exhibit a limited capacity to repair damaged cells or regenerate new ones. In contrast, animals ranging from flies to fishes and even some mammals like the spiny mouse exhibit innate abilities to regenerate their kidney cells following injury. Now, a recent study has illuminated how the Mexican salamander, Ambystoma mexicanum, most commonly known as the axolotl, possesses a kidney with remarkable similarity to humans, which can robustly regenerate following acute chemical damage. These discoveries position the axolotl as a new model that can be used to advance our understanding about the fundamental mechanisms of kidney regeneration.

Differential effects of 3,5-T2 and T3 on the gill regeneration and metamorphosis of the Ambystoma mexicanum (axolotl).

Thyroid hormones (THs) regulate tissue remodeling processes during early- and post-embryonic stages in vertebrates. The Mexican axolotl (Ambystoma mexicanum) is a neotenic species that has lost the ability to undergo metamorphosis; however, it can be artificially induced by exogenous administration of thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). Another TH derivative with demonstrative biological effects in fish and mammals is 3,5-diiodo-L-thyronine (3,5-T2). Because the effects of this bioactive TH remains unexplored in other vertebrates, we hypothesized that it could be biologically active in amphibians and, therefore, could induce metamorphosis in axolotl. We performed a 3,5-T2 treatment by immersion and observed that the secondary gills were retracted, similar to the onset stage phenotype; however, tissue regeneration was observed after treatment withdrawal. In contrast, T4 and T3 immersion equimolar treatments as well as a four-fold increase in 3,5-T2 concentration triggered complete metamorphosis. To identify the possible molecular mechanisms that could explain the contrasting reversible or irreversible effects of 3,5-T2 and T3 upon gill retraction, we performed a transcriptomic analysis of differential expression genes in the gills of control, 3,5-T2-treated, and T3-treated axolotls. We found that both THs modify gene expression patterns. T3 regulates 10 times more genes than 3,5-T2, suggesting that the latter has a lower affinity for TH receptors (TRs) or that these hormones could act through different TR isoforms. However, both TH treatments regulated different gene sets known to participate in tissue development and cell cycle processes. In conclusion, 3,5-T2 is a bioactive iodothyronine that promoted partial gill retraction but induced full metamorphosis in higher concentrations. Differential effects on gill retraction after 3,5,-T2 or T3 treatment could be explained by the activation of different clusters of genes related with apoptosis, regeneration, and proliferation; in addition, these effects could be initially mediated by TRs that are expressed in gills. This study showed, for the first time, the 3,5,-T2 bioactivity in a neotenic amphibian.

RNA localization during early development of the axolotl.

The asymmetric localization of biomolecules is critical for body plan development. One of the most popular model organisms for early embryogenesis studies is Xenopus laevis but there is a lack of information in other animal species. Here, we compared the early development of two amphibian species-the frog X. laevis and the axolotl Ambystoma mexicanum. This study aimed to identify asymmetrically localized RNAs along the animal-vegetal axis during the early development of A. mexicanum. For that purpose, we performed spatial transcriptome-wide analysis at low resolution, which revealed dynamic changes along the animal-vegetal axis classified into the following categories: profile alteration, de novo synthesis and degradation. Surprisingly, our results showed that many of the vegetally localized genes, which are important for germ cell development, are degraded during early development. Furthermore, we assessed the motif presence in UTRs of degraded mRNAs and revealed the enrichment of several motifs in RNAs of germ cell markers. Our results suggest novel reorganization of the transcriptome during embryogenesis of A. mexicanum to converge to the similar developmental pattern as the X. laevis.