Transcriptomics of A. mexicanum Regeneration: A Comparative Study Reveals Key Proteins And Pathways for Limb Regeneration.
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ABSTRACT:
ORGANISM(S): Ambystoma mexicanum
PROVIDER: GSE237864 | GEO | 2024/11/27
REPOSITORIES: GEO
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Project description: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.
2023-05-08 | GSE185593 | GEO
Project description:Humans and other tetrapods are considered to require apical-ectodermal-ridge, AER, cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, the primary regeneration model organism, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first established that axolotls contain cells with AER characteristics. Surprisingly, further analyses and spatial transcriptomics revealed that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a novel program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.
2023-09-20 | GSE243225 | GEO
Project description:Human diseases like oral cancer or injuries may cause irreversible substantial jaw defects. Axolotls are of special interest in regenerative maxillofacial research due to their unique ability to completely renew amputated jaws. Here we performed differential gene expression analyses based on RNA-seq for regenerating jaw tissue of the Mexican axolotl (Ambystoma mexicanum) to identify key players in this regenerative process. Within differentially expressed genes (p<0.001) we use enrichment and network analyses to identify increased expression of structural components crucial for tissue repair (Thbs3, Thbs4 and Col3a1) as well as for extracellular matrix structure (further collagens and Matn4). Genes of enzymatic function (AldoA, AldoC, Eno1, Ckm and Ak6) as well as muscle components (Atp2A1, Pvalb, Tpm3) are down-regulated. This first RNA-seq study on axolotl jaw tissue can help to elucidate the general molecular mechanisms underlying mandible regeneration and offer a chance for new regenerative strategies focusing on morphologically and functionally adequate replacement of these complex tissues.
2018-12-01 | GSE95355 | GEO
Project description: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 similarity 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 pacing. 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.
2024-08-29 | GSE271056 | GEO
Project description:Salamanders have the remarkable ability to functionally regenerate after spinal cord transection. In response to injury, GFAP+ glial cells in the axolotl spinal cord proliferate and migrate to replace the missing neural tube and create a permissive environment for axon regeneration. Molecular pathways that regulate the pro-regenerative axolotl glial cell response are poorly understood. Here we show axolotl glial cells up-regulate AP-1cFos/JunB after injury, which promotes a pro-regenerative glial cell response. Axolotl glial cells directly repress c-Jun expression via up-regulation of miR-200a. Inhibition of miR-200a during regeneration causes defects in axonal regrowth and transcriptomic analysis revealed that miR-200a inhibition leads to differential regulation of genes involved with reactive gliosis, the glial scar, ECM remodeling and axon guidance. This work identifies a novel role for miR-200a in inhibiting reactive gliosis in glial cell in axolotl during spinal cord regeneration
2019-03-19 | GSE122939 | GEO
Project description:Salamanders, such as the Mexican axolotl, are some of the few vertebrates fortunate in their ability to regenerate diverse structures after injury. Unlike mammals they are able to regenerate a fully functional spinal cord after injury. However, the early signals required to initiate a pro-regenerative response after spinal cord injury is not well understood. To address this question we developed a spinal cord injury model in axolotls and used in vivo imaging of ion sensitive dyes and determined that spinal cord injury induces a rapid and dynamic change in the resting membrane potential of ependymoglial cells. Prolonged depolarization of ependymoglial cells after injury inhibits glial cell proliferation and subsequent axon regeneration. Using transcriptional profiling we identified c-Fos as a key voltage sensitive early response gene that is expressed specifically in the ependymoglial cells after injury. This data establishes that dynamic changes in the membrane potential after injury are essential for regulating the specific spatiotemporal expression of c-Fos that is critical for promoting faithful spinal cord regeneration in axolotl. All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2â3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed. Experimental Design: Ivermectin injection Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate.
2016-07-05 | E-GEOD-71934 | biostudies-arrayexpress
Project description:Identifying the genetic program that induces limb regeneration in salamanders is an important resource for regenerative medicine, which currently lacks tools to promote regeneration of functional body structures. The genetic network underlying limb regeneration has been elusive due to the complexity of the injury response that occurs concomitant to blastema formation. Here we performed parallel expression profile time courses of non-regenerative lateral wounds versus amputated limbs in axolotl. We show that limb regeneration occurs in three distinguishable phases--early wound healing followed by a transition phase leading to establishment of the limb development program. By focusing on the transition phase, we identified 93 strictly regeneration-associated genes involved in oxidative stress response, chromatin modification, epithelial development and limb development. The specific expression of the genes was confirmed by in situ hybridization. Regeneration-specific expression databases are critical resources for understanding how regeneration-relevant phenotypes can be induced from adult cells Regeneration of the axolotl forelimb lower arm was compared with the healing of a deep lateral injury in a high density timecourse (uncut, 3h, 6h, 9h, 12h, 24h, 36h, 52h, 72h, 120h, 168h, 288h and 528h after injury). Three independent biological replicates were performed using separate cluches of animals. Amputated and lateral wound samples were made as matched contralateral samples of four pooled animals per timepoint.
2013-01-01 | E-GEOD-36451 | biostudies-arrayexpress
Project description:Amphibians such as the 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), a cell-cycle-regulated protein that prevents cells from entering mitosis prematurely, is of great interest for it is highly upregulated in the limb blastema of axolotls, but its expression level remains unchanged in the fibroblastema of postmetamorphic frogs. Yet, its role in regeneration competent context in Xenopus has not been fully analyzed. Here we show that Evi5 is also upregulated in Xenopus tadpoles after limb and tail amputation, as it is in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs wound healing and blastema formation in limbs and tails in both axolotls and Xenopus tadpoles, suggesting a conserved function for Evi5 in regeneration. Using skin punch as a healing model we show that Evi5 is also involved in cell migration during wound healing. RNA-sequencing analysis shows that in addition to reduced signaling of Lepr, Pdgfa, Gdf5, evi5 Mo also downregulate lysine demethylases kdm6b and kdm7a, which are also required for limb regeneration. Thus, our results demonstrate that Evi5 plays a critical role in the regeneration of multiple systems in amphibians.
2022-11-24 | GSE218034 | GEO
Project description:While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair. We used microarray analysis to determine the gene expression changes that take place during scar free wound healing in aquatic and terrestrial axolotl salamanders. Epidermal tissue was harvested using a 4mm biopsy punch. Two wounds were made along the flank and posterior to the forelimbs. Harvested epidermis was pooled for each animal. Four biological replicates were collected from uninjured epidermis (D0) and at 1, 3, and 7 days post injury.
2012-01-31 | E-GEOD-35255 | biostudies-arrayexpress
Project description:Limb regeneration, while observed lifelong in salamanders, is restricted to pre-metamorphic stages in Xenopus laevis frogs. After amputation, post-metamorphic frogs form a blastema that grows only an unsegmented cartilage rod. Whether this loss is due to systemic factors such as the immune system or due to an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that, as in axolotl, connective tissue (CT) cells form the post-metamorphic frog blastema. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells fully dedifferentiate and integrate to form lineages including cartilage in the developing limb. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT but not to developing cartilage. Further, using single-cell RNA-seq analysis we find that the embryonic and the adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.
2021-05-24 | GSE165901 | GEO