Evolutionarily divergent mTOR remodels translatome for tissue regeneration.

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

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.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

Embryonic Tissue and Blastema Transplantations.

Embryo grafts have been an experimental pillar in developmental biology, and particularly, in amphibian biology. Grafts have been essential in constructing fate maps of different cell populations and migratory patterns. Likewise, autografts and allografts in older larvae or adult salamanders have been widely used to disentangle mechanisms of regeneration. The combination of transgenesis and grafting has widened even more the application of this technique.In this chapter, we provide a detailed protocol for embryo transplants in the axolotl (Ambystoma mexicanum ). The location and stages to label connective tissue, muscle, or blood vessels in the limb and blood cells in the whole animal. However, the potential of embryo transplants is enormous and impossible to cover in one chapter. Furthermore, we provide a protocol for blastema transplantation as an example of allograft in older larvae.

Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration.

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.

Single-cell Stereo-seq reveals induced progenitor cells involved in axolotl brain regeneration.

The molecular mechanism underlying brain regeneration in vertebrates remains elusive. We performed spatial enhanced resolution omics sequencing (Stereo-seq) to capture spatially resolved single-cell transcriptomes of axolotl telencephalon sections during development and regeneration. Annotated cell types exhibited distinct spatial distribution, molecular features, and functions. We identified an injury-induced ependymoglial cell cluster at the wound site as a progenitor cell population for the potential replenishment of lost neurons, through a cell state transition process resembling neurogenesis during development. Transcriptome comparisons indicated that these induced cells may originate from local resident ependymoglial cells. We further uncovered spatially defined neurons at the lesion site that may regress to an immature neuron-like state. Our work establishes spatial transcriptome profiles of an anamniote tetrapod brain and decodes potential neurogenesis from ependymoglial cells for development and regeneration, thus providing mechanistic insights into vertebrate brain regeneration.

Single-cell analyses of axolotl telencephalon organization, neurogenesis, and regeneration.

Salamanders are tetrapod models to study brain organization and regeneration; however, the identity and evolutionary conservation of brain cell types are largely unknown. We delineated the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-cell genomic profiling. We identified glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved γ-aminobutyric acid-releasing (GABAergic) neuron classes. We inferred transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific neurogenesis and unraveled conserved differentiation signatures. After brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield insights into the organization, evolution, and regeneration of a tetrapod nervous system.

Axolotl: A resourceful vertebrate model for regeneration and beyond.

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

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.

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.

The Tale-Tell Heart: Evolutionary tetrapod shift from aquatic to terrestrial life-style reflected in heart changes in axolotl (Ambystoma mexicanum).

BACKGROUND: During amphibian metamorphosis, the crucial moment lies in the rearrangement of the heart, reflecting the changes in circulatory demands. However, little is known about the exact shifts linked with this rearrangement. Here, we demonstrate such myocardial changes in axolotl (Ambystoma mexicanum) from the morphological and physiological point of view. RESULTS: Micro-CT and histological analysis showed changes in ventricular trabeculae organization, completion of the atrial septum and its connection to the atrioventricular valve. Based on Myosin Heavy Chain and Smooth Muscle Actin expression we distinguished metamorphosis-induced changes in myocardial differentiation at the ventricular trabeculae and atrioventricular canal. Using optical mapping, faster speed of conduction through the atrioventricular canal was demonstrated in metamorphic animals. No differences between the groups were observed in the heart rates, ventricular activation times, and activation patterns. CONCLUSIONS: Transition from aquatic to terrestrial life-style is reflected in the heart morphology and function. Rebuilding of the axolotl heart during metamorphosis was connected with reorganization of ventricular trabeculae, completion of the atrial septum and its connection to the atrioventricular valve, and acceleration of AV conduction.

Neural control of growth and size in the axolotl limb regenerate.

The mechanisms that regulate growth and size of the regenerating limb in tetrapods such as the Mexican axolotl are unknown. Upon the completion of the developmental stages of regeneration, when the regenerative organ known as the blastema completes patterning and differentiation, the limb regenerate is proportionally small in size. It then undergoes a phase of regeneration that we have called the 'tiny-limb' stage, which is defined by rapid growth until the regenerate reaches the proportionally appropriate size. In the current study we have characterized this growth and have found that signaling from the limb nerves is required for its maintenance. Using the regenerative assay known as the accessory limb model (ALM), we have found that growth and size of the limb positively correlates with nerve abundance. We have additionally developed a new regenerative assay called the neural modified-ALM (NM-ALM), which decouples the source of the nerves from the regenerating host environment. Using the NM-ALM we discovered that non-neural extrinsic factors from differently sized host animals do not play a prominent role in determining the size of the regenerating limb. We have also discovered that the regulation of limb size is not autonomously regulated by the limb nerves. Together, these observations show that the limb nerves provide essential cues to regulate ontogenetic allometric growth and the final size of the regenerating limb.

Humans' ability to regrow lost or damaged body parts is relatively limited, but some animals, such as the axolotl (a Mexican salamander), can regenerate complex body parts, like legs, many times over their lives. Studying regeneration in these animals could help researchers enhance humans' abilities to heal. One way to do this is using the Accessory Limb Model (ALM), where scientists wound an axolotl's leg, and study the additional leg that grows from the wound. The first stage of limb regeneration creates a new leg that has the right structure and shape. The new leg is very small so the next phase involves growing the leg until its size matches the rest of the animal. This phase must be controlled so that the limb stops growing when it reaches the right size, but how this regulation works is unclear. Previous research suggests that the number of nerves in the new leg could be important. Wells et al. used a ALM to study how the size of regenerating limbs is controlled. They found that changing the number of nerves connected to the new leg altered its size, with more nerves leading to a larger leg. Next, Wells et al. created a system that used transplanted nerve bundles of different sizes to grow new legs in different sized axolotls. This showed that the size of the resulting leg is controlled by the number of nerves connecting it to the CNS. Wells et al. also showed that nerves can only control regeneration if they remain connected to the central nervous system. These results explain how size is controlled during limb regeneration in axolotls, highlighting the fact that regrowth is directly controlled by the number of nerves connected to a regenerating leg. Much more work is needed to reveal the details of this process and the signals nerves use to control growth. It will also be important to determine whether this control system is exclusive to axolotls, or whether other animals also use it.

Lung injury in axolotl salamanders induces an organ-wide proliferation response.

BACKGROUND: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. RESULTS: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. CONCLUSION: Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.

Optogenetic manipulation of cellular communication using engineered myosin motors.

Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.

Chemical genetics of regeneration: Contrasting temporal effects of CoCl2 on axolotl tail regeneration.

BACKGROUND: Histone deacetylases (HDACs) regulate transcriptional responses to injury stimuli that are critical for successful tissue regeneration. Previously we showed that HDAC inhibitor romidepsin potently inhibits axolotl tail regeneration when applied for only 1-minute postamputation (MPA). RESULTS: Here we tested CoCl2, a chemical that induces hypoxia and cellular stress, for potential to reverse romidepsin inhibition of tail regeneration. Partial rescue of regeneration was observed among embryos co-treated with romidepsin and CoCl2 for 1 MPA, however, extending the CoCl2 dosage window either inhibited regeneration (CoCl2 :0 to 30 MPA) or was lethal (CoCl2 :0 to 24 hours postamputation; HPA). CoCl2 :0 to 30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, while CoCl2 treatment of non-amputated embryos or CoCl2 :60 to 90 MPA treatment after re-epithelialization did not inhibit tail regeneration. CoCl2 -romidepsin:1 MPA treatment partially restored expression of transcription factors that are typical of appendage regeneration, while CoCl2 :0 to 30 MPA significantly increased expression of genes associated with cell stress and inflammation. Additional experiments showed that CoCl2 :0 to 1 MPA and CoCl2 :0 to 30 MPA significantly increased levels of glutathione and reactive oxygen species, respectively. CONCLUSION: Our study identifies a temporal window from tail amputation to re-epithelialization, within which injury activated cells are highly sensitive to CoCl2 perturbation of redox homeostasis.

Single-cell RNA-seq reveals novel mitochondria-related musculoskeletal cell populations during adult axolotl limb regeneration process.

While the capacity to regenerate tissues or limbs is limited in mammals, including humans, axolotls are able to regrow entire limbs and major organs after incurring a wound. The wound blastema has been extensively studied in limb regeneration. However, due to the inadequate characterization of ECM and cell subpopulations involved in the regeneration process, the discovery of the key drivers for human limb regeneration remains unknown. In this study, we applied large-scale single-cell RNA sequencing to classify cells throughout the adult axolotl limb regeneration process, uncovering a novel regeneration-specific mitochondria-related cluster supporting regeneration through energy providing and the ECM secretion (COL2+) cluster contributing to regeneration through cell-cell interactions signals. We also discovered the dedifferentiation and re-differentiation of the COL1+/COL2+ cellular subpopulation and exposed a COL2-mitochondria subcluster supporting the musculoskeletal system regeneration. On the basis of these findings, we reconstructed the dynamic single-cell transcriptome of adult axolotl limb regenerative process, and identified the novel regenerative mitochondria-related musculoskeletal populations, which yielded deeper insights into the crucial interactions between cell clusters within the regenerative microenvironment.

A histological study of normal and pathological limb regeneration in the Mexican axolotl Ambystoma mexicanum.

Salamanders show unparalleled capacities of tissue regeneration amongst tetrapods (four-legged vertebrates), being able to repair and renew lost or damage body parts, such as tails, jaws, and limbs in a seemingly perfect fashion. Despite countless studies on axolotl (Ambystoma mexicanum) regeneration, only a few studies have thus far compared gross morphological and histological features of the original and regenerated limb skeleton. Therein, most studies have focused on nerves or muscles, while even fewer have provided detailed information about bones and cartilage. This study compares skeletal tissue structures of original and regenerated limbs with respect to tissue level histology. Histological serial sections of 55 axolotl larvae were generated, including 29 limbs that were severed by conspecifics, and 26 that were subject to targeted amputations. Amputations were executed in several larval stages (48, 52, and 53) and at different limb positions (humeral midshaft, above the mesopod). In addition, 3D reconstructions were prepared based on X-ray microtomography scans. The results demonstrate that regenerated forelimbs show a diversity of limb and digit abnormalities as a result of imperfect regeneration. Furthermore, abnormalities were more severe and more frequent in regenerated forelimbs caused by natural bites as compared with regenerated forelimbs after amputation. The results indicate that abnormalities occur frequently after regeneration in larval axolotls contradicting the notion of regeneration generally resulting in perfect limbs.

DNA repair during regeneration in Ambystoma mexicanum.

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

A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

Neural regulation in tooth regeneration of Ambystoma mexicanum.

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