Transgenic animals have been indispensable in elucidating and deciphering mechanisms underlying various biological phenomena. In regeneration, transgenic animals expressing fluorescent protein genes have been crucial for identifying the source cells for regeneration and the mechanism of blastema formation. Animals are usually generated by manipulating their genome using various techniques at/in one cell embryo/fertilized egg stage. Here, we describe the generation of germline transgenic axolotls (Ambystoma mexicanum) using the I-SceI meganuclease and Tol2 transposase.

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

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The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

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.

The aim of this paper is to assemble a significant amount of information on Ambystoma mexicanum, the axolotl salamander, to assist in the basic knowledge needed to raise, breed, and study most aspects of axolotl biology. It is important to understand the basic biology of the axolotl in order to make informed decisions on their proper care and use in experiments. Therefore, we will provide necessary information to the non-herpetologist that will assist in their study of this unique and fascinating animal. We also aim to provide a resource on the general anatomy, behavior, and experimental tips specific to the Mexican axolotl that will be of use to most axolotl laboratories. Axolotls have been actively researched since the 1860s, giving testament to their relatively straightforward maintenance and their versatility as an animal model for development and regeneration. Interest in using the axolotl in laboratory research has grown tremendously over the past decade, so dedicated resources to support the study of this species are needed and encouraged.

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration.

The laboratory axolotl (Ambystoma mexicanum) is widely used in biological research. Recent advancements in genetic and molecular toolkits are greatly accelerating the work using axolotl, especially in the area of tissue regeneration. At this juncture, there is a critical need to establish gene and transgenic nomenclature to ensure uniformity in axolotl research. Here, we propose guidelines for genetic nomenclature when working with the axolotl.

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.

Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV).

Gill regeneration has not been well studied compared to regeneration of other appendages, such as limb and tail regeneration. Here, we focused on axolotl gill regeneration and found that Fgf- and Bmp-signaling are involved in their gill regeneration mechanism. Axolotls have three pairs of gill rami, and each gill ramus has multiple gill filaments. The gills consist of mesenchyme rich in extracellular matrix and epidermis. The gill nerves are supplied from the trigeminal ganglia located in the head. Denervation resulted in no gill regeneration responses. Nerves and gills express Bmp and Fgf genes, and treating animals with Fgf- and Bmp-signaling inhibitors results in phenotypes similar to those seen in denervated gills. Inducing an accessory appendage is a standard assay in amphibian regeneration research. In our study, an accessory gill could be induced by lateral wounding, suggesting that thin axon fibers and mesenchymal Fgfs and Bmps contributed to the induction of the accessory structure. Such accessory gill induction was inhibited by the denervation. Exogenous Fgf2+Fgf8+Bmp7, which have been determined to function as a regeneration inducer in urodele amphibians, could compensate for the effects denervation has on accessory blastema formation. Our findings suggest that regeneration of appendages in axolotls is regulated by common Fgf- and Bmp-signaling cascades.

In Tennessee, populations of the state endangered Streamside Salamander (Ambystoma barbouri) are in decline as their distribution lies mostly within rapidly developing areas in the Nashville Basin. Information regarding the partitioning of genetic variation among populations of A. barbouri and the taxonomic status of these populations relative to northern populations and their congener, the Small-mouthed Salamander (A. texanum), have important implications for management and conservation of this species. Here we combined mitochondrial sequencing and genome-wide single nucleotide polymorphism (SNP) data generated using Genotyping-by-Sequencing (GBS) to investigate patterns of genetic variation within Tennessee populations of A. barbouri, to assess their relationship to populations in Kentucky, and to examine their phylogenetic relationship to the closely related A. texanum. Results from phylogenetic reconstructions reveal a complex history of Tennessee A. barbouri populations with regards to northern populations, unisexual A. barbouri, and A. texanum. Patterns of mitochondrial sequence variation suggest that A. barbouri may have originated within Tennessee and expanded north multiple times into Kentucky, Ohio, Indiana, and West Virginia. Phylogenetic reconstructions based on genome-wide SNP data contradict results based on mitochondrial DNA and correspond to geographic and taxonomic boundaries. Variation in allele frequencies at SNP genotypes, as identified by multivariate analyses and Bayesian assignment tests, identified three evolutionary significant units (ESUs) for A. barbouri within Tennessee. Collectively, these results emphasize the need for prioritizing conservation needs for Tennessee populations of A. barbouri to ensure the long-term persistence of this species.

Vertebrate eye formation requires coordinated inductive interactions between different embryonic tissue layers, first described in amphibians. A network of transcription factors and signaling molecules controls these steps, with mutations causing severe ocular, neuronal, and craniofacial defects. In eyeless mutant axolotls, eye morphogenesis arrests at the optic vesicle stage, before lens induction, and development of ventral forebrain structures is disrupted.

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.

Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers.

The age of unisexual salamanders of the genus Ambystoma is contentious. Recent and ancient evolutionary histories of unisexual Ambystoma were proposed by a few separate studies that constructed phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other studies showing that unisexual Ambystoma represent the most ancient unisexual vertebrates, a recent study by Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago.

Sex pheromones have been shown to constitute a crucial aspect of salamander reproduction. Until now, courtship pheromones of Salamandridae and Plethodontidae have been intensively studied, but information on chemical communication in other urodelan families is essentially lacking. The axolotl (Ambystoma mexicanum, Ambystomatidae) has a courtship display that suggests a key role for chemical communication in the orchestration of its sexual behavior, but no sex pheromones have yet been characterized from this species. Here we combined whole transcriptome analyses of the male cloaca with proteomic analyses of water in which axolotls were allowed to court to show that male axolotls secrete multiple ca. 20 kDa glycosylated sodefrin precursor-like factor (SPF) proteins during courtship. In combination with phylogenetic analyses, our data show that the male cloaca essentially secretes a courtship-specific clade of SPF proteins that is orthologous to salamandrid courtship pheromones. In addition, we identified an SPF protein for which no orthologs have been described from other salamanders so far. Overall, our study advocates a central role for SPF proteins during the courtship display of axolotls and adds knowledge on pheromone use in a previously unexplored deep evolutionary branch of salamander evolution.

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.