A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration.

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.

Regulation of stem cell identity by miR-200a during spinal cord regeneration.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.

Making a new limb out of old cells: exploring endogenous cell reprogramming and its role during limb regeneration.

Cellular reprogramming is characterized by the induced dedifferentiation of mature cells into a more plastic and potent state. This process can occur through artificial reprogramming manipulations in the laboratory such as nuclear reprogramming and induced pluripotent stem cell (iPSC) generation, and endogenously in vivo during amphibian limb regeneration. In amphibians such as the Mexican axolotl, a regeneration permissive environment is formed by nerve-dependent signaling in the wounded limb tissue. When exposed to these signals, limb connective tissue cells dedifferentiate into a limb progenitor-like state. This state allows the cells to acquire new pattern information, a property called positional plasticity. Here, we review our current understanding of endogenous reprogramming and why it is important for successful regeneration. We will also explore how naturally induced dedifferentiation and plasticity were leveraged to study how the missing pattern is established in the regenerating limb tissue.

FGF signaling induces the regeneration of collagen fiber structure during skin wound healing in axolotls.

Axolotls have been considered to be able to regenerate their skin completely. Our recent study updated this theory with the finding that the lattice structure of dermal collagen fibers was not fully regenerated after skin injury. We also discovered that nerves induce the regeneration of collagen fibers. The mechanism of collagen fiber regeneration remains unknown, however. In this study, we focused on the structure of collagen fibers with collagen braiding cells, and cell origin in axolotl skin regeneration. In the wounded dermis, cells involved in skin repair/regeneration were derived from both the surrounding dermis and the subcutaneous tissue. Regardless of cell origin, cells acquired the proper cell morphology to braid collagen fiber with nerve presence. We also found that FGF signaling could substitute for the nerve roles in the conversion of subcutaneous fibroblasts to lattice-shaped dermal fibroblasts. Our findings contribute to the elucidation of the fundamental mechanisms of true skin regeneration and provide useful insights for pioneering new skin treatments.

Tenascin-C-enriched regeneration-specific extracellular matrix guarantees superior muscle regeneration in Ambystoma mexicanum.

Severe muscle injury causes distress and difficulty in humans. Studying the high regenerative ability of the axolotls may provide hints for the development of an effective treatment for severe injuries to muscle tissue. Here, we examined the regenerative process in response to a muscle injury in axolotls. We found that axolotls are capable of complete regeneration in response to a partial muscle resection called volumetric muscle loss (VML), which mammals cannot perfectly regenerate. We investigated the mechanisms underlying this high regenerative capacity in response to VML, focusing on the migration of muscle satellite cells and the extracellular matrix (ECM) formed during VML injury. Axolotls form tenascin-C (TN-C)-enriched ECM after VML injury. This TN-C-enriched ECM promotes the satellite cell migration. We confirmed the importance of TN-C in successful axolotl muscle regeneration by creating TN-C mutant animals. Our results suggest that the maintenance of a TN-C-enriched ECM environment after muscle injury promotes the release of muscle satellite cells and supports eventually high muscle regenerative capacity. In the future, better muscle regeneration may be achieved in mammals through the maintenance of TN-C expression.

Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl.

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.

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.

Temporal microbiome changes in axolotl limb regeneration: Stage-specific restructuring of bacterial and fungal communities with a Flavobacterium bloom during blastema proliferation.

The intricate relationship between regeneration and microbiota has recently gained attention, spanning diverse model organisms. Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite its significance, a noticeable gap exists in understanding the interplay between axolotl regeneration and its microbiome. Here, we analyse in depth bacterial 16S rRNA amplicon dataset that we reported before as data resource and profile fungal community by sequencing ITS amplicons at the critical stages of limb regeneration (0-1-4-7-30-60 days post amputation, 'dpa'). Results reveal a decline in richness and evenness in the course of limb regeneration, with bacterial community richness recovering beyond 30 dpa unlike fungi community. Beta diversity analysis reveals precise restructuring of the bacterial community along the three phases of limb regeneration, contrasting with less congruent changes in the fungal community. Temporal dynamics of the bacterial community highlight prevalent anaerobic bacteria in initiation phase and Flavobacterium bloom in the early phase correlating with limb blastema proliferation. Predicted functional analysis mirrors these shifts, emphasising a transition from amino acid metabolism to lipid metabolism control. Fungal communities shift from Blastomycota to Ascomycota dominance in the late regeneration stage. Our findings provide ecologically relevant insights into stage specific role of microbiome contributions to axolotl limb regeneration.

Host Species and Environment Shape the Skin Microbiota of Mexican Axolotls.

Skin microbiomes in amphibians are complex systems that can be influenced by biotic and abiotic factors. In this study, we examined the effect of host species and environmental conditions on the skin bacterial and fungal microbiota of four obligate paedomorphic salamander species, commonly known as axolotls (Ambystoma andersoni, A. dumerilii, A. mexicanum, and A. taylori), all of them endemic to the Trans-Mexican Volcanic Belt. We found that despite their permanent aquatic lifestyle, these species present a host-specific skin microbiota that is distinct from aquatic communities. We identified skin-associated taxa that were unique to each host species and that differentiated axolotl species based on alpha and beta diversity metrics. Moreover, we identified a set of microbial taxa that were shared across hosts with high relative abundances across skin samples. Specifically, bacterial communities were dominated by Burkholderiales and Pseudomonadales bacterial orders and Capnodiales and Pleosporales fungal orders. Host species and environmental variables collectively explained more microbial composition variation in bacteria (R2 = 0.46) in comparison to fungi (R2 = 0.2). Our results contribute to a better understanding of the factors shaping the diversity and composition of skin microbial communities in Ambystoma. Additional studies are needed to disentangle the effects of specific host associated and environmental factors that could influence the skin microbiome of these endangered species.

Hemodynamics During Development and Postnatal Life.

A well-developed heart is essential for embryonic survival. There are constant interactions between cardiac tissue motion and blood flow, which determine the heart shape itself. Hemodynamic forces are a powerful stimulus for cardiac growth and differentiation. Therefore, it is particularly interesting to investigate how the blood flows through the heart and how hemodynamics is linked to a particular species and its development, including human. The appropriate patterns and magnitude of hemodynamic stresses are necessary for the proper formation of cardiac structures, and hemodynamic perturbations have been found to cause malformations via identifiable mechanobiological molecular pathways. There are significant differences in cardiac hemodynamics among vertebrate species, which go hand in hand with the presence of specific anatomical structures. However, strong similarities during development suggest a common pattern for cardiac hemodynamics in human adults. In the human fetal heart, hemodynamic abnormalities during gestation are known to progress to congenital heart malformations by birth. In this chapter, we discuss the current state of the knowledge of the prenatal cardiac hemodynamics, as discovered through small and large animal models, as well as from clinical investigations, with parallels gathered from the poikilotherm vertebrates that emulate some hemodynamically significant human congenital heart diseases.

The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci.

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

BMP signaling is essential for sustaining proximo-distal progression in regenerating axolotl limbs.

Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is downregulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development.

Two is not necessarily better than one: A double lens in a pre-metamorphic adult axolotl (Ambystoma mexicanum).

An 8-year-old female pre-metamorphic axolotl (Ambystoma mexicanum) was examined for a suspected anterior lens luxation. Slit-lamp biomicroscopy revealed two lens-like structures in the anterior chamber of the right eye (OD), each with cataractous change. Ultrasound biomicroscopy and optical coherence tomography (OCT) were performed without sedation, and revealed small lenticular structures each with distinct nuclei and cortices. Although a distinct connection of the two lenticular structures could not be definitively ruled out, the structures appeared separate. Each of the lenticular structures was closely associated with its respective iris leaflet. This report demonstrates application of advanced imaging for diagnostic use in axolotl ophthalmology, showing that imaging of the lens can be performed without sedation, topical anesthetic, nor contact gel with high diagnostic quality. Although two distinct lenses were diagnosed with no historical evidence of trauma, the small sizes of each lenticular structure, with no detectable connection between them, are suggestive of a possible regenerative abnormality. This report opens discussion for the regenerative capabilities of the pre-metamorphic adult axolotl and possible implementations of their use in regenerative medicine research for the development of future therapies.

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

BACKGROUND: 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. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: 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.

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.

Tgf-ß superfamily and limb regeneration: Tgf-ß to start and Bmp to end.

Axolotls represent a popular model to study how nature solved the problem of regenerating lost appendages in tetrapods. Our work over many years focused on trying to understand how these animals can achieve such a feat and not end up with a scarred up stump. The Tgf-ß superfamily represents an interesting family to target since they are involved in wound healing in adults and pattern formation during development. This family is large and comprises Tgf-ß, Bmps, activins and GDFs. In this review, we present work from us and others on Tgf-ß & Bmps and highlight interesting observations between these two sub-families. Tgf-ß is important for the preparation phase of regeneration and Bmps for the redevelopment phase and they do not overlap with one another. We present novel data showing that the Tgf-ß non-canonical pathway is also not active during redevelopment. Finally, we propose a molecular model to explain how Tgf-ß and Bmps maintain distinct windows of expression during regeneration in axolotls.

Evidence of requirement for homologous-mediated DNA repair during Ambystoma mexicanum limb regeneration.

BACKGROUND: Limb regeneration in the axolotl is achieved by epimorphosis, thus depending on the blastema formation, a mass of progenitor cells capable of proliferating and differentiating to recover all lost structures functionally. During regeneration, the blastema cells accelerate the cell cycle and duplicate its genome, which is inherently difficult to replicate because of its length and composition, thus being prone to suffer double-strand breaks. RESULTS: We identified and characterized two remarkable components of the homologous recombination repair pathway (Amex.RAD51 and Amex.MRE11), which were heterologously expressed, biochemically characterized, and inhibited by specific chemicals. These same inhibitors were applied at different time points after amputation to study their effects during limb regeneration. We observed an increase in cellular senescent accompanied by a slight delay in regeneration at 28 days postamputation regenerated tissues; moreover, inhibitors caused a rise in the double-strand break signaling as a response to the inhibition of the repair mechanisms. CONCLUSIONS: We confirmed the participation and importance of homologous recombination during limb regeneration. The chemical inhibition induces double-strand breaks that lead to DNA damage associated senescence, or in an alternatively way, this damage could be possibly repaired by a different DNA repair pathway, permitting proper regeneration and avoiding senescence.

Lmx1b activation in axolotl limb regeneration.

BACKGROUND: Axolotls can regenerate their limbs. In their limb regeneration process, developmental genes are re-expressed and reorganize the developmental axes, in which the position-specific genes are properly re-expressed. However, how such position specificity is reorganized in the regeneration processes has not been clarified. To address this issue, we focused on the reactivation process of Lmx1b, which determines the limb dorsal identity in many animals. RESULTS: Here, we show that Lmx1b expression is maintained in the dorsal skin before amputation and is activated after amputation. Furthermore, we demonstrate that only cells located in the dorsal side prior to limb amputation could reactivate Lmx1b after limb amputation. We also found that Lmx1b activation was achieved by nerve presence. The nerve factors, BMP2+FGF2+FGF8 (B2FF), consistently reactivate Lmx1b when applied to the dorsal skin. CONCLUSIONS: These results imply that the retained Lmx1b expression in the intact skin plays a role in positional memory, which instruct cells about the spatial positioning before amputation. This memory is reactivated by nerves or nerve factors that can trigger the entire limb regeneration process. Our findings highlight the role of nerves in amphibian limb regeneration, including both the initiation of limb regeneration and the reactivation of position-specific gene expression.

Distinct toll-like receptor signaling in the salamander response to tissue damage.

BACKGROUND: Efficient wound healing or pathogen clearance both rely on balanced inflammatory responses. Inflammation is essential for effective innate immune-cell recruitment; however, excessive inflammation will result in local tissue destruction, pathogen egress, and ineffective pathogen clearance. Sterile and nonsterile inflammation operate with competing functional priorities but share common receptors and overlapping signal transduction pathways. In regenerative organisms such as the salamander, whole limbs can be replaced after amputation while exposed to a nonsterile environment. In mammals, exposure to sterile-injury Damage Associated Molecular Patterns (DAMPS) alters innate immune-cell responsiveness to secondary Pathogen Associated Molecular Pattern (PAMP) exposure. RESULTS: Using new phospho-flow cytometry techniques to measure signaling in individual cell subsets we compared mouse to salamander inflammation. These studies demonstrated evolutionarily conserved responses to PAMP ligands through toll-like receptors (TLRs) but identified key differences in response to DAMP ligands. Co-exposure of macrophages to DAMPs/PAMPs suppressed MAPK signaling in mammals, but not salamanders, which activate sustained MAPK stimulation in the presence of endogenous DAMPS. CONCLUSIONS: These results reveal an alternative signal transduction network compatible with regeneration that may ultimately lead to the promotion of enhanced tissue repair in mammals.