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.
Project description:Axolotl limb regeneration proceeds through the formation of a blastema, a mound of progenitor cells that accumulate at the end of the amputated stump. These progenitor cells expand and later undergo patterning to regenerate the missing limb, restoring both form and function. A subset of cells within the blastema become senescent, a state of permanent growth arrest. Here, we address the functional relevance of cellular senescence to axolotl limb regeneration, through a combination of gain- and loss-of-function assays. Using transcriptomic analyses on in vitro and in vivo senescent cells, we gain insights into the basis of the senescent phenotype, cell-cycle arrest, and molecular mediators involved in axolotl regeneration at the molecular level.
Project description:Axolotl limb regeneration proceeds through the formation of a blastema, a mound of progenitor cells that accumulate at the end of the amputated stump. These progenitor cells expand and later undergo patterning to regenerate the missing limb, restoring both form and function. A subset of cells within the blastema become senescent, a state of permanent growth arrest. Here, we address the functional relevance of cellular senescence to axolotl limb regeneration, through a combination of gain- and loss-of-function assays. Using transcriptomic analyses on in vitro and in vivo senescent cells, we gain insights into the basis of the senescent phenotype, cell-cycle arrest, and molecular mediators involved in axolotl regeneration at the molecular level.
Project description:Axolotl limb regeneration proceeds through the formation of a blastema, a mound of progenitor cells that accumulate at the end of the amputated stump. These progenitor cells expand and later undergo patterning to regenerate the missing limb, restoring both form and function. A subset of cells within the blastema become senescent, a state of permanent growth arrest. Here, we address the functional relevance of cellular senescence to axolotl limb regeneration, through a combination of gain- and loss-of-function assays. Using transcriptomic analyses on in vitro and in vivo senescent cells, we gain insights into the basis of the senescent phenotype, cell-cycle arrest, and molecular mediators involved in axolotl regeneration at the molecular level.
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.
Project description:Discovery of genes driving axolotl limb regeneration has been challenging due to limited genomic resources. We assembled 42 RNA-Seq samples totaling approximately 1.3 billion 100 base paired-end reads using Trinity (Grabherr M.G. et al, Nature Biotechnology, 2011; Haas B.J. et al, Nature Protocols, 2013): https://github.com/trinityrnaseq/trinityrnaseq/wiki). We created a transcriptome with complete sequence information for most axolotl genes, identified transcriptional profiles that distinguish blastemas from differentiated limb tissues, and uncovered functional roles for cirbp and kazald1 in limb regeneration.
Project description:Regeneration of complex multi-tissue structures, such as limbs, requires the coordinated effort of multiple cell types. In axolotl limb regeneration, the wound epidermis and blastema have been extensively studied via histology, grafting, and bulk-tissue RNA-sequencing. However, studying the contributions of these tissues is hindered due to limited information regarding the molecular identity of the cell types in regenerating limbs. By performing unbiased single-cell RNA-sequencing on over 25,000 cells from axolotl limbs, we identify a plethora of cellular diversity within epidermal, mesenchymal, and hematopoietic lineages in homeostatic and regenerating limbs. We identify regeneration-induced genes, develop putative trajectories for blastema cell differentiation, and propose the molecular identity and origin of fibroblast-derived blastema progenitor cells residing in homeostatic limbs. This work will enable application of molecular techniques to assess the contribution of these populations to limb regeneration. It will also facilitate work aimed at identifying transcripts and cells critical for limb regeneration.
Project description:Here we report the results of a robust microarray experiment that examined the first 28 days of axolotl forelimb regeneration. At each of 20 post-amputation time points, we estimated gene expression for 10 replicate RNA samples that were isolated from 1m of heterogeneous tissue collected from the distal limb tip. The distal 1.0mm of heterogeneous tissue from regenerating limb tip were removed and used for RNA extraction. 10 samples were collected for each of 20 time points.
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