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Previous studies have reported that amputation invokes body-wide responses in regenerative organisms, but most have not examined the implications of these changes beyond the region of tissue regrowth. Specifically, long-range epidermal responses to amputation are largely uncharacterized, with research on amputation-induced epidermal responses in regenerative organisms traditionally being restricted to the wound site. Here, we investigate the effect of amputation on long-range epidermal permeability in two evolutionarily distant, regenerative organisms: axolotls and planarians. We find that amputation triggers a long-range increase in epidermal permeability in axolotls, accompanied by a long-range epidermal downregulation in MAPK signaling. Additionally, we provide functional evidence that pharmacologically inhibiting MAPK signaling in regenerating planarians increases long-range epidermal permeability. These findings advance our knowledge of body-wide changes due to amputation in regenerative organisms and warrant further study on whether epidermal permeability dysregulation in the context of amputation may lead to pathology in both regenerative and non-regenerative organisms.

RESUMEN

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.

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Appendage regeneration is not a simple task. The animal must harness all of its energy and resources to orchestrate perhaps one of the most complicated events since its development. Balancing the immune response, wound healing, proliferation, patterning and differentiation is an elegant job, and how some animals achieve that still leaves researchers enchanted today. In this work, we review some of the molecular aspects of regeneration, with a focus on the axolotl, the champion of tetrapod limb regeneration, and the mouse, an excellent mammalian model for digit tip regeneration. Advances in molecular and genomic tools have enabled the discovery of exciting fundamental features of limb regeneration. Integrating the data from different animal systems will be crucial to understanding the common requirements of successful appendage regeneration and places for flexibility. The combination of these efforts is paving the way to grasping how good regenerators respond to the loss of body parts, how these mechanisms might compare in modest regenerators, and, ultimately, in developing approaches for improving regenerative outcomes in humans.

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Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.

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Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.

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Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl's ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls' ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.