Search

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue‐patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re‐induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signaling center that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal–ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridization and mathematical modeling, we first quantified the behaviors of dorsal–ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labeled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signaling center in the axolotl spinal cord. [ABSTRACT FROM AUTHOR]

Previous studies have implicated epithelial–mesenchymal transition (EMT) in salamander limb regeneration. In this review, we describe putative roles for EMT during each stage of limb regeneration in axolotls and other salamanders. We hypothesize that EMT and EMT‐like gene expression programs may regulate three main cellular processes during limb regeneration: (1) keratinocyte migration during wound closure; (2) transient invasion of the stump by epithelial cells undergoing EMT; and (3) use of EMT‐like programs by non‐epithelial blastemal progenitor cells to escape the confines of their niches. Finally, we propose nontraditional roles for EMT during limb regeneration that warrant further investigation, including alternative EMT regulators, stem cell activation, and fibrosis induced by aberrant EMT. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that periderm (the outer ectodermal layer) in zebrafish partially expands into the mouth and pharyngeal pouches, but does not reach the medial endoderm, where the pharyngeal teeth develop. Instead, periderm-like cells, arising independently from the outer periderm, cover prospective tooth-forming epithelia and are crucial for tooth germ initiation. Here we test the hypothesis that restricted expansion of periderm is a teleost-specific character possibly related to the derived way of early embryonic development. To this end, we performed lineage tracing of the periderm in a non-teleost actinopterygian species possessing pharyngeal teeth, the sterlet sturgeon (Acipenser ruthenus), and a sarcopterygian species lacking pharyngeal teeth, the axolotl (Ambystoma mexicanum). Results: In sturgeon, a stratified ectoderm is firmly established at the end of gastrulation, with minimally a basal ectodermal layer and a surface layer that can be homologized to a periderm. Periderm expands to a limited extent into the mouth and remains restricted to the distal parts of the pouches. It does not reach the medial pharyngeal endoderm, where pharyngeal teeth are located. Thus, periderm in sturgeon covers prospective odontogenic epithelium in the jaw region (oral teeth) but not in the pharyngeal region. In axolotl, like in sturgeon, periderm expansion in the oropharynx is restricted to the distal parts of the opening pouches. Oral teeth in axolotl develop long before mouth opening and possible expansion of the periderm into the mouth cavity. Conclusions: Restricted periderm expansion into the oropharynx appears to be an ancestral feature for osteichthyans, as it is found in sturgeon, zebrafish and axolotl. Periderm behavior does not correlate with presence or absence of oral or pharyngeal teeth, whose induction may depend on 'ectodermalized' endoderm. It is proposed that periderm assists in lumenization of the pouches to create an open gill slit. Comparison of basal and advanced actinopterygians with sarcopterygians (axolotl) shows that different trajectories of embryonic development converge on similar dynamics of the periderm: a restricted expansion into the mouth and prospective gill slits. [ABSTRACT FROM AUTHOR]

Axolotls are known for their remarkable regeneration ability. Exploring their transcriptome provides insight into regenerative mechanisms. However, the current annotation of the axolotl transcriptome is limited, leaving the role of unannotated transcripts in regeneration unknown. To discourse this challenge, we exploited long-read sequencing technology, which enables direct observation of full-length RNA transcripts, greatly enhancing the coverage and accuracy of axolotl transcriptome annotation. By utilizing this method, we identified 222 novel gene loci and 4775 novel transcripts, which were quantified using short-read sequencing data. Through the inclusive analysis, we discovered novel homologs, potential functional proteins, noncoding RNAs, and alternative splicing events in key regeneration pathways. In particular, we identified novel transcripts with high protein-coding potential implicated in cell cycle regulation and musculoskeletal development, and regeneration were identified. Interestingly, alternative splice variants were also detected across diverse pathways critical to regeneration. This specifies that these novel transcripts potentially play vital roles underpinning the robust regenerative capacities of axolotls. Single-cell transcriptomic analysis further revealed these isoforms to predominantly exist in axolotl limb chondrocytes and mature tissue cell populations. Overall, the findings significantly advanced consideration of the axolotl transcriptome and provided a new perspective for understanding the mechanisms of regenerative abilities of axolotls.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The axolotl (Ambystoma mexicanum) is renowned for its remarkable regenerative capabilities, which are not diminished by the transition from a neotenic to a metamorphic state. This study explored the microbiome dynamics in axolotl limb regeneration by examining the microbial communities present in neotenic and metamorphic axolotls at two critical stages of limb regeneration: pre-amputation and during blastema formation. Utilizing 16S rRNA amplicon sequencing, we investigated the variations in microbiome profiles associated with different developmental and regenerative states. Our findings reveal a distinct separation in the microbiome profiles of neotenic and metamorphic samples, with a clear demarcation in microbial composition at both the phylum and genus levels. In neotenic 0DPA samples, Proteobacteria and Firmicutes were the most abundant, whereas in neotenic 7DPA samples, Proteobacteria and Bacteroidetes dominated. Conversely, metamorphic samples displayed a higher abundance of Firmicutes and Bacteroidetes at 0DPA and Proteobacteria and Firmicutes at 7DPA. Alpha and beta diversity analyses, along with dendrogram construction, demonstrated significant variations within and between the sample groups, suggesting a strong influence of both developmental stage and regenerative state on the microbiome. Notably, Flavobacterium and Undibacterium emerged as distinctive microbial entities in neotenic 7DPA samples, highlighting potential key players in the microbial ecology of regeneration. These findings suggest that the axolotl's microbiome is dynamically responsive to blastema formation, and they underscore the potential influence of microbial communities on the regeneration process. This study lays the groundwork for future research into the mechanisms by which the microbiome may modulate regenerative capacity. [ABSTRACT FROM AUTHOR]

Neoteny is a developmental strategy wherein an organism reaches sexual maturity without associated adult characteristics. In salamanders, neoteny takes the form of individuals retaining aquatic larval characteristics such as external gills upon maturation. Mole salamanders (Ambystoma) occupy a wide range of habitats and areas across the North American continent, and display examples of non‐neotenic, facultatively neotenic and obligate neotenic species, providing high variation for investigating the factors influencing the evolution of neoteny. Here, we use phylogenetic comparative methods to test existing hypotheses that neoteny is associated with elevational and latitudinal distribution, cave‐associated isolation, and hybridisation‐related polyploidy. We also test if neoteny influences the diversity of habitats a species can occupy, since the restriction to an aquatic life should constrain the availability of different niches. We find that neoteny tends to occur in a narrow latitudinal band between 20–30° North, with particularly narrow latitudinal ranges for obligate compared to facultative neotenic species (16–52° North). We also find that facultatively neotenic species occur at elevations more than twice as high as other species on average, and that species with a higher frequency of neoteny typically have lower habitat diversity. Our results suggest that evolutionary transitions between non‐neotenic and facultative neoteny states occur at relatively high and approximately equal rates. Moreover, we estimate that obligate neoteny cannot evolve directly from non‐neotenic species (and vice versa), such that facultative neoteny acts as an evolutionary 'stepping stone' to and from obligate neoteny. However, our transition rate estimates suggest that obligate neoteny is lost >4‐times faster than it evolves, partly explaining the rarity of obligate species. These results support the hypothesis that low latitudes favour the evolution of neoteny, presumably linked to more stable (aquatic) environments due to reduced seasonality, but once evolved it may constrain the diversity of habitats. [ABSTRACT FROM AUTHOR]

Objective: The present study focused on the effect of different lipid levels on growth (weight and length) and survival in juvenile Ambystoma mexicanum. Design/methodology/approach: Four diets with the same 45% protein level and different lipid levels: 6, 8, 10 and 12% were tested for a period of 81 days. For the preparation of the diets, two key ingredients were used, such as fishmeal and fish oil, these as protein base and lipid source. The experiment consisted of placing six organisms per experimental unit in tubs with 40 L of water for a period of 81 days; period during which four biometrics were performed, the organisms were fed every 48 hours to the weight of their biomass. The digestibility of the diets, initial and final height, initial and final weight, weight gained, weight gained per day, specific growth rate, survival, Fulton index and protein efficiency rate were recorded. Results: At the end of the experiment, significant differences (p>0.05) were observed in the growth and survival of the axolotls in the diets of 8, 10 and 12% lipids. Limitations on study/implications: A wider range of lipid levels could not be tested, due to the number of individuals available for the experiment. Findings/conclusions: According to the data obtained in this study, it is recommended to include a level of 45% protein and 8% lipids in the diets of juvenile Ambystoma mexicanum, for their better development and nutrition. [ABSTRACT FROM AUTHOR]

Neurodegenerative proteinopathies such as Alzheimer's Disease are characterized by abnormal protein aggregation and neurodegeneration. Neuroresilience or regenerative strategies to prevent neurodegeneration, preserve function, or restore lost neurons may have the potential to combat human proteinopathies; however, the adult human brain possesses a limited capacity to replace lost neurons. In contrast, axolotls (Ambystoma mexicanum) show robust brain regeneration. To determine whether axolotls may help identify potential neuroresilience or regenerative strategies in humans, we first interrogated whether axolotls express putative proteins homologous to human proteins associated with neurodegenerative diseases. We compared the homology between human and axolotl proteins implicated in human proteinopathies and found that axolotls encode proteins highly similar to human microtubule-binding protein tau (tau), amyloid precursor protein (APP), and β-secretase 1 (BACE1), which are critically involved in human proteinopathies like Alzheimer's Disease. We then tested monoclonal Tau and BACE1 antibodies previously used in human and rodent neurodegenerative disease studies using immunohistochemistry and western blotting to validate the homology for these proteins. These studies suggest that axolotls may prove useful in studying the role of these proteins in disease within the context of neuroresilience and repair. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Amphibians are vitally important in biomedical research and conservation. Preserving germplasm materials (e.g., sperm, egg, embryos) in cryopreserved form can reduce the costs and risks associated with maintenance of live organisms. The goal of this work was to establish an initial practical sperm cryopreservation pathway for axolotls (Ambystoma mexicanum) to provide a foundation for development of germplasm repositories. The specific objectives were to (1) establish an efficient approach for sperm collection and quality evaluation; (2) evaluate the effects of osmotic pressure of extender solutions on sperm quality during refrigerated storage; (3) evaluate the effects of cryoprotectants on post-thaw sperm quality; (4) evaluate the feasibility of fertilization with thawed sperm; and (5) establish a cryopreservation pathway by integrating the findings into a comprehensive process flow map. With this approach, axolotl offspring were produced with cryopreserved sperm for the first time. Future research can build from this work with the goal of developing a germplasm repository based on community-level integration at the Ambystoma Genetic Stock Center. The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25–800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks' Balanced Salt Solution (HBSS) at osmolalities of 100–600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7–8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Axolotl muscle regeneration ability is superior to that of mammals. • The presence of regeneration-promoting ECM contributes to higher muscle regeneration. • Tenascin-C is the component consisting of the regeneration-promoting ECM. • Loss of Tenascin-C impairs the axolotl muscle regeneration ability. [ABSTRACT FROM AUTHOR]

Background: Traumatic spinal cord injury (SCI) is a disabling condition that affects millions of people around the world. Currently, no clinical treatment can restore spinal cord function. Comparison of molecular responses in regenerating to non-regenerating vertebrates can shed light on neural restoration. The axolotl (Ambystoma mexicanum) is an amphibian that regenerates regions of the brain or spinal cord after damage. Methods: In this study, we compared the transcriptomes after SCI at acute (1–2 days after SCI) and sub-acute (6–7 days post-SCI) periods through the analysis of RNA-seq public datasets from axolotl and non-regenerating rodents. Results: Genes related to wound healing and immune responses were upregulated in axolotls, rats, and mice after SCI; however, the immune-related processes were more prevalent in rodents. In the acute phase of SCI in the axolotl, the molecular pathways and genes associated with early development were upregulated, while processes related to neuronal function were downregulated. Importantly, the downregulation of processes related to sensorial and motor functions was observed only in rodents. This analysis also revealed that genes related to pluripotency, cytoskeleton rearrangement, and transposable elements (e.g., Sox2, Krt5, and LOC100130764) were among the most upregulated in the axolotl. Finally, gene regulatory networks in axolotls revealed the early activation of genes related to neurogenesis, including Atf3/4 and Foxa2. Conclusions: Immune-related processes are upregulated shortly after SCI in axolotls and rodents; however, a strong immune response is more noticeable in rodents. Genes related to early development and neurogenesis are upregulated beginning in the acute stage of SCI in axolotls, while the loss of motor and sensory functions is detected only in rodents during the sub-acute period of SCI. The approach employed in this study might be useful for designing and establishing regenerative therapies after SCI in mammals, including humans. [ABSTRACT FROM AUTHOR]

Abstract Neoteny is a developmental strategy wherein an organism reaches sexual maturity without associated adult characteristics. In salamanders, neoteny takes the form of individuals retaining aquatic larval characteristics such as external gills upon maturation. Mole salamanders (Ambystoma) occupy a wide range of habitats and areas across the North American continent, and display examples of non‐neotenic, facultatively neotenic and obligate neotenic species, providing high variation for investigating the factors influencing the evolution of neoteny. Here, we use phylogenetic comparative methods to test existing hypotheses that neoteny is associated with elevational and latitudinal distribution, cave‐associated isolation, and hybridisation‐related polyploidy. We also test if neoteny influences the diversity of habitats a species can occupy, since the restriction to an aquatic life should constrain the availability of different niches. We find that neoteny tends to occur in a narrow latitudinal band between 20–30° North, with particularly narrow latitudinal ranges for obligate compared to facultative neotenic species (16–52° North). We also find that facultatively neotenic species occur at elevations more than twice as high as other species on average, and that species with a higher frequency of neoteny typically have lower habitat diversity. Our results suggest that evolutionary transitions between non‐neotenic and facultative neoteny states occur at relatively high and approximately equal rates. Moreover, we estimate that obligate neoteny cannot evolve directly from non‐neotenic species (and vice versa), such that facultative neoteny acts as an evolutionary ‘stepping stone’ to and from obligate neoteny. However, our transition rate estimates suggest that obligate neoteny is lost >4‐times faster than it evolves, partly explaining the rarity of obligate species. These results support the hypothesis that low latitudes favour the evolution of neoteny, presumably linked to more stable (aquatic) environments due to reduced seasonality, but once evolved it may constrain the diversity of habitats.

Amphibians such as salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5) is of great interest because its expression is highly upregulated in the limb blastema of axolotls, but remains unchanged in the fibroblastema of post-metamorphic frogs. Yet, its role in regeneration-competent contexts in Xenopus has not been fully analyzed. Here we show that Evi5 is upregulated in Xenopus tadpoles after limb and tail amputation, as in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs limb development and limb blastema formation in Xenopus tadpoles. Mechanistically, we show that Evi5 knockdown significantly reduces proliferation of limb blastema cells and causes apoptosis, blocking the formation of regeneration blastema. RNA-sequencing analysis reveals that in addition to reduced PDGFα and TGFβ signaling pathways that are required for regeneration, evi5 Mo downregulates lysine demethylases Kdm6b and Kdm7a. And knockdown of Kdm6b or Kdm7a causes defective limb regeneration. Evi5 knockdown also impedes tail regeneration in Xenopus tadpoles and axolotl larvae, suggesting a conserved function of Evi5 in appendage regeneration. Thus, our results demonstrate that Evi5 plays a critical role in appendage regeneration in amphibians.

Circular RNAs (circRNAs) are of relevance to regenerative medicine and play crucial roles in post-transcriptional and translational regulation of biological processes. circRNAs are a class of RNA molecules that are formed through a unique splicing process, resulting in a covalently closed-loop structure. Recent advancements in RNA sequencing technologies and specialized computational tools have facilitated the identification and functional characterization of circRNAs. These molecules are known to exhibit stability, developmental regulation, and specific expression patterns in different tissues and cell types across various organisms. However, our understanding of circRNA expression and putative function in model organisms for regeneration is limited. In this context, this study reports, for the first time, on the repertoire of circRNAs in axolotl, a widely used model organism for regeneration. We generated RNA-seq data from intact limb, wound, and blastema tissues of axolotl during limb regeneration. The analysis revealed the presence of 35,956 putative axolotl circRNAs, among which 5331 unique circRNAs exhibited orthology with human circRNAs. In silico data analysis underlined the potential roles of axolotl circRNAs in cell cycle, cell death, and cell senescence-related pathways during limb regeneration, suggesting the participation of circRNAs in regulation of diverse functions pertinent to regenerative medicine. These new observations help advance our understanding of the dynamic landscape of axolotl circRNAs, and by extension, inform future regenerative medicine research and innovation that harness this model organism. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Yap1 expression in neotenic axolotls' blastema was higher than in metamorphic ones. • The effect of Yap1 KD on axolotl limb regeneration was explored. • Depletion of Yap1 during limb regeneration results in bone defects. • Altered BPs upon Yap1 inhibition were revealed using proteomics. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Collagen-producing cells in the axolotl dermis have lattice-patterned filopodia. • Dermal cells do not restore lattice morphology after skin injury. • Subcutaneous cells contribute to dermal reformation regardless of nerve presence. • Nerve factor FGFs induce the lattice formation of subcutaneous fibroblasts. [ABSTRACT FROM AUTHOR]

Intussusception diagnosis and surgical management in axolotls (Ambystoma mexicanum) is poorly documented. A client-owned, 5-year-old, sexually intact, female axolotl was presented for hyporexia of 4-week duration associated with regurgitation after feedings. Clinical examination showed lethargy, weight loss, and firm tissue at coelomic palpation. Coelomic ultrasonography was consistent with an intestinal intussusception. An exploratory coeliotomy was performed, followed by an intestinal resection and anastomosis of a thickened portion of intestinal loop. Following surgical excision of the invaginated intestinal loops, anorexia was not resolved, and the axolotl died 4 days later. A necropsy revealed a serofibrinous coelomitis. Histopathology confirmed the presence of an obstructive mass in the resected portion of the intestines. This report describes an intussusception diagnosis and attempted treatment in an axolotl. Ultrasonography in axolotls with nonspecific gastrointestinal symptoms is recommended for evaluation of the coelomic organs. [ABSTRACT FROM AUTHOR]

Limb regeneration is the outcome of a complex sequence of events that are mediated by interactions between cells derived from the tissues of the amputated stump. Early in regeneration, these interactions are mediated by growth factor/morphogen signaling associated with nerves and the wound epithelium. One shared property of these proregenerative signaling molecules is that their activity is dependent on interactions with sulfated glycosaminoglycans (GAGs), heparan sulfate proteoglycan (HSPG) in particular, in the extracellular matrix (ECM). We hypothesized that there are cells in the axolotl that synthesize specific HSPGs that control growth factor signaling in time and space. In this study we have identified a subpopulation of cells within the ECM of axolotl skin that express high levels of sulfated GAGs on their cell surface. These cells are dispersed in a grid-like pattern throughout the dermis as well as the loose connective tissues that surround the tissues of the limb. These cells alter their morphology during regeneration, and are candidates for being a subpopulation of connective tissue cells that function as the cells required for pattern-formation during regeneration. Given their high level of HSPG expression, their stellate morphology, and their distribution throughout the loose connective tissues, we refer to these as the positional information GRID (Groups that are Regenerative, Interspersed and Dendritic) cells. In addition, we have identified cells that stain for high levels of expression of sulfated GAGs in mouse limb connective tissue that could have an equivalent function to GRID cells in the axolotl. The identification of GRID cells may have important implications for work in the area of Regenerative Engineering.

Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP+ tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Reported neoplasia in amphibians is rare with axolotls (Ambystoma mexicanum) and African clawed frogs (Xenopus laevis) being overrepresented. Reported neoplasia in axolotls include chromatophoromas, teratomas, and neuroblastomas. A 3-year-old intact female axolotl was presented with a pigmented bilobed right proximal tail mass and a vascular left proximal tail mass. The patient was anesthetized with alfaxalone via immersion bath and subcutaneous injection, and the tail was amputated proximal to the masses. The axolotl recovered uneventfully and was discharged with oral medications. Histopathology of the tail masses revealed incomplete excision of presumptive iridophoromas (a subset of chromatophoroma). Complete tail regeneration occurred after 7 months with no gross reoccurrence of the masses. The patient presented 9 months post-operatively with a 3-week history of decreased appetite. Coelomic ultrasound revealed a vascularized coelomic mass caudal to the liver. Euthanasia was performed and necropsy revealed presumptive metastasis of the chromatophoromas into the spleen and kidneys. Chromatophoromas with potential for metastasis should be considered in axolotls with cutaneous masses. [ABSTRACT FROM AUTHOR]

Introduction: Little is known about how the newly regenerated limb tissues in the Mexican axolotl seamlessly integrate with the remaining stump tissues to form a functional structure, and why this doesn't occur in some regenerative scenarios. In this study, we evaluate the phenomenological and transcriptional characteristics associated with integration failure in ectopic limb structures generated by treating anterior-located ectopic blastemas with Retinoic Acid (RA) and focusing on the “bulbus mass” tissue that forms between the ectopic limb and the host site. We additionally test the hypothesis that the posterior portion of the limb base contains anterior positional identities.Methods: The positional identity of the bulbus mass was evaluated by assaying regenerative competency, the ability to induce new pattern in the Accessory Limb Model (ALM) assay, and by using qRTPCR to quantify the relative expression of patterning genes as the bulbus mass deintegrates from the host site. We additionally use the ALM and qRTPCR to analyze the distribution of anterior and posterior positional identities along the proximal/distal limb axis of uninjured and regenerating limbs.Results: The bulbus mass regenerates limb structures with decreased complexity when amputated and is able to induce complex ectopic limb structure only when grafted into posterior-located ALMs. Expressional analysis shows significant differences in FGF8, BMP2, TBX5, Chrdl1, HoxA9, and HoxA11 expression between the bulbus mass and the host site when deintegration is occuring. Grafts of posterior skin from the distal limb regions into posterior ALMs at the base of the limb induce ectopic limb structures. Proximally-located blastemas express significantly less HoxA13 and Ptch1, and significantly more Alx4 and Grem1 than distally located blastemas.Discussion: These findings show that the bulbus mass has an anterior-limb identity and that the expression of limb patterning genes is mismatched between the bulbus mass and the host limb. Our findings additionally show that anterior positional information is more abundant at the limb base, and that anterior patterning genes are more abundantly expressed in proximally located blastemas compared to blastemas in the more distal regions of the limb. These experiments provide valuable insight into the underlying causes of integration failure and further map the distribution of positional identities in the mature limb.

The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25–800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks’ Balanced Salt Solution (HBSS) at osmolalities of 100–600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7–8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications.

The tetrapod salamander species axolotl (Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the axolotl brain. Here, we establish and optimize an electroporationbased method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC (Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of axolotl brain regeneration. [ABSTRACT FROM AUTHOR]

We report on the discovery of a population related to the Blunt-headed Salamander (Ambystoma amblycephalum), a micro-endemic axolotl from Mexico scientifically confirmed only once since its original description in 1940, and now presumably extinct. In 2018, paedomorphic and metamorphosed adults, as well as clutches and larvae, were found in a cattle pond at Nahuatzen, Michoacán state, Mexico, ~60 km away from the type locality (Tacícuaro). Morphometric comparisons suggested high similarity with the type series of A. amblycephalum, while mitochondrial DNA barcoding (16S and control region) revealed close (but imperfect) matching to a reference sequence. We gathered data on life history and ecology of this population, which could be the only extant relic of A. amblycephalum. Its highly limited distribution and presumably low population density are hallmarks for a high risk of extinction, and alarms on the critical situation of many micro-endemic salamanders of Mexico, hence calling for immediate conservation actions. [ABSTRACT FROM AUTHOR]

Amphibians such as salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5) is of great interest because its expression is highly upregulated in the limb blastema of axolotls, but remains unchanged in the fibroblastema of post-metamorphic frogs. Yet, its role in regeneration-competent contexts in Xenopus has not been fully analyzed. Here we show that Evi5 is upregulated in Xenopus tadpoles after limb and tail amputation, as in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs limb development and limb blastema formation in Xenopus tadpoles. Mechanistically, we show that Evi5 knockdown significantly reduces proliferation of limb blastema cells and causes apoptosis, blocking the formation of regeneration blastema. RNA-sequencing analysis reveals that in addition to reduced PDGFα and TGFβ signaling pathways that are required for regeneration, evi5 Mo downregulates lysine demethylases Kdm6b and Kdm7a. And knockdown of Kdm6b or Kdm7a causes defective limb regeneration. Evi5 knockdown also impedes tail regeneration in Xenopus tadpoles and axolotl larvae, suggesting a conserved function of Evi5 in appendage regeneration. Thus, our results demonstrate that Evi5 plays a critical role in appendage regeneration in amphibians.

Regeneration of a complex appendage structure such as limb requires upstream and downstream coordination of multiple types of cells. Given type of cell may sit at higher upstream position to control the activities of other cells. Muscles are one of the major cell masses in limbs. However, the subtle functional relationship between muscle and other cells in vertebrate complex tissue regeneration are still not well established. Here, we use Pax7 mutant axolotls, in which the limb muscle is developmentally lost, to investigate limb regeneration in the absence of skeletal muscle. We find that the pattern of regenerated limbs is relative normal in Pax7 mutants compared to the controls, but the joint is malformed in the Pax7 mutants. Lack of muscles do not affect the early regeneration responses, specifically the recruitment of macrophages to the wound, as well as the proliferation of fibroblasts, another major population in limbs. Furthermore, using single cell RNA-sequencing, we show that, other than muscle lineage that is mostly missing in Pax7 mutants, the composition and the status of other cell types in completely regenerated limbs of Pax7 mutants are similar to that in the controls. Our study reveals skeletal muscle is barely required for the guidance of other cells, as well the patterning in complex tissue regeneration in axolotls, and provides refined views of the roles of muscle cell in vertebrate appendage regeneration. [ABSTRACT FROM AUTHOR]

In his essay "Why Look at Animals?," John Berger states that in the first encounter between a human and an animal, the latter's eyes are attentive and wary. The animal looks at the human from the abyss of incomprehension. The human becomes aware of himself by looking back at the animal, who is the radical Other. If the abyss of otherness between humans can be bridged through language, this is impossible between the human and the animal. The gaze is the only possibility for the human to "descend" to the animal. Julio Cortázar wrote "Axolotl," which tells the story of a man who, fascinated by the axolotls, goes to the Jardin des Plantes aquarium in Paris every day to observe them and ends up becoming one of them. Already in the first paragraph of the story, the character-narrator states: "Now I am an axolotl." Then he adds: "His eyes above all obsessed me. Next to them in the other aquariums, various fish showed me the simple stupidity of their beautiful eyes like ours. The eyes of the axolotls told me of the presence of a different life, of another way of looking." The narrator manages to transcend the barrier of "space and time" through the gaze and, finally, becomes an axolotl. [ABSTRACT FROM AUTHOR]

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. Key Findings: The mechanism of cell spatial recognition in limb regeneration is unknown.We investigated the activation process of Lmx1b, which is important to establish dorsal identity.Lmx1b is activated only in cells that were located in the dorsal region before amputation.Lmx1b is activated by nerves or by the nerve factors, BMP2+FGF2+FGF8.We suggest that the positional memory of the cells is maintained as faint expressions of position‐specific genes and that the nerve factors are required for its activation. [ABSTRACT FROM AUTHOR]

Axolotl (Ambystoma mexicanum) limb regeneration begins with blastemas of various sizes, in contrast to the limb developmental process. Despite this size variation, normal limb morphology, consistent with a limb stump size, is regenerated. This outcome suggests the existence of underlying scale-invariant mechanisms. To identify such mechanisms, we examined the allometric relationships between blastema size, and Sonic Hedgehog (Shh) and Fibroblast Growth Factor 8 (Fgf8) expression patterns against limb stump size. We found that all factors showed allometric rather than isometric scaling; specifically, their relative sizes decrease with an increase in limb stump size. However, the ratio of Shh/Fgf8 signaling dominant region was nearly constant, independent of blastema/body size. Furthermore, the relative spatial patterns of cell density and proliferation activity, and the relative position of first digit formation were scale invariant in the summed Shh/Fgf8 crosstalk region. This scale-invariant nature may underlie the morphogenesis of normal limbs from different sizes of blastemas., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The mandible plays an essential part in human life and, thus, defects in this structure can dramatically impair the quality of life in patients. Axolotls, unlike humans, are capable of regenerating their lower jaws; however, the underlying mechanisms and their similarities to those in limb regeneration are unknown. In this work, we used morphological, histological and transcriptomic approaches to analyze the regeneration of lateral resection defects in the axolotl mandible. We found that this structure can regenerate all missing tissues in 90 days through gap minimization, blastema formation and, finally, tissue growth, differentiation and integration. Moreover, transcriptomic comparisons of regenerating mandibles and limbs showed that they share molecular phases of regeneration, that these similarities peak during blastema stages and that mandible regeneration occurs at a slower pace. Altogether, our study demonstrates the existence of a shared regenerative program used in two different regenerating body structures with different embryonic origins in the axolotl and contributes to our understanding of the minimum requirements for a successful regeneration in vertebrates, bringing us closer to understand similar lesions in human mandibles., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.

Objective: To determine the protein nutritional requirements in juvenile axolotls, for this purpose, four isocaloric diets (8 % lipids) with 30, 35, 30 and 45 % protein were prepared. Design/methodology/approach: 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 hours with 4% of the weight of the biomass of organisms per experimental reservoir. Four biometries 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 (p> 0.05) in the growth and survival of the axolotls, the diet with 45% protein showed the best growth results. Limitations/Implications: No more protein levels could be tested, due to the number of organisms available for bioassays. Findings/conclusions: Diets for A. mexicanum containing 45% protein level promote good development and survival. This allows for improved cultivation and management plans for the species. [ABSTRACT FROM AUTHOR]

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. Key Findings: A. mexicanum possesses true homologs for Rad51 and Mre11.B02 and Mirin chemicals inhibit Amex.RAD51 and Amex.MRE11 respectively.B02 and Mirin provoke an increase in DNA damage during regeneration.B02 produces an increase in the number of senescent cells during regeneration. [ABSTRACT FROM AUTHOR]

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. Key Findings: The juvenile appendicular skeleton is formed mostly by Sox9/Col1a2 cartilage cells, transitioning to a slow and life‐long ossification that starts around sexual maturation.Axolotl growth is biphasic, defined by an explosive growth of axolotls in approximately the first 20 months of their life, after which growth velocity dramatically diminishes, however never reaching zero.A hybrid population of hypertrophic chondrocytes, expressing the chondrogenic marker SOX9 and the osteoblast marker OCN, resides next to the ossification center.Col1a2‐descendants contribute to bone and adipocytes during ossification. [ABSTRACT FROM AUTHOR]

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‐β 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‐β, Bmps, activins and GDFs. In this review, we present work from us and others on Tgf‐β & Bmps and highlight interesting observations between these two sub‐families. Tgf‐β 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‐β non‐canonical pathway is also not active during redevelopment. Finally, we propose a molecular model to explain how Tgf‐β and Bmps maintain distinct windows of expression during regeneration in axolotls. Key Findings: Tgf‐β and Bmp control different phases of limb regeneration.Tgf‐β superfamily is essential throughout regeneration.Limb regeneration has two distinct phases controlled by different signaling pathways. [ABSTRACT FROM AUTHOR]