Allometry in limb regeneration and scale-invariant patterning as the basis of normal morphogenesis from different sizes of blastemas.

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.

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.