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Therapeutic implementation of human limb regeneration is a daring aim. Studying species that can regrow their lost appendages provides clues on how such a feat can be achieved in mammals. One of the unique features of regeneration-competent species lies in their ability to seal the amputation plane with a scar-free wound epithelium. Subsequently, this wound epithelium advances and becomes a specialized wound epidermis (WE) which is hypothesized to be the essential component of regenerative success. Recently, the WE and specialized WE terminologies have been used interchangeably. However, these tissues were historically separated, and contemporary limb regeneration studies have provided critical new information which allows us to distinguish them. Here, I will summarize tissue-level observations and recently identified cell types of WE and their specialized forms in different regeneration models.
Fig. 1. a Schematic to generate a multi-species limb atlas using publicly available single-cell RNA-Seq (scRNA-Seq) datasets. b UMAP plot of Seurat-integrated multi-species limb atlas. Individual datasets from each species with different developmental stages are integrated. Dots are colored by cell identities. c UMAP plot of the ectodermal lineage. Dots are colored by cell identities. d UMAP plot of species contribution to the AER cluster. Dots are colored by species. e Dot plot showing AER marker expressions in AER cells from different species. The dot color indicates the mean expression that was normalized to the max of each dataset and to the max of each gene; the dot size represents the percentage of cells with non-zero expression. f Heatmap showing the MetaNeighbor score for pair-wise similarities of basal ectoderm and AER clusters. X- and Y-axes indicate species and developmental stages. Asterisks (*) denote the pairs with scores above 0.9. Source data provided as a Source data file. g Heatmap showing signaling ligands gene set enrichment analysis scores for AER, and non-AER basal ectoderm clusters. The basal ectoderm represents the transcriptome-wide most similar population to the AER, and is used for comparison. Colored dots in the Y-axis indicate different species. Source data provided as a Source data file. h Single optical section of z-stacks of confocal images of Stage 46 axolotl forelimb buds stained for Dr999-Pmt21178 (referred to as Dr999) mRNA via hybridization-chain-reaction (HCR). Different z-stacks were shown from left to right, representing different levels of the dorsal-ventral axis. (Top) Gray, Hoechst; Bottom Gray, Dr999 mRNA. Scale bar: 100 μm. i Max-projection confocal image of Stage 53 axolotl forelimbdigit tips stained for Dr999 mRNA via HCR. Green, Dr999 mRNA; Gray, Hoechst. Scale bar: 250 μm. j Zoomed single optical section image of the axolotl limb bud from (h) stained for Dr999 mRNA. Red arrows show Dr999+ squamous cells, and yellow arrows show outer layer peridermal cells. The basement membrane is labeled with a dashed line. Green, Dr999 mRNA; Gray, Hoechst. Scale bar: 10 μm.
FIGURE 1. The wound epidermis (WE) and the specialized wound epidermis form in a step-wise manner during amphibian limb regeneration. Limb regeneration is initiated with amputation in (top) salamanders and (bottom) tadpoles. The remaining stump epidermal cells migrate to the amputation plane (purple arrows) and form the WE (light blue). Then the WE becomes the specialized wound epidermis [apical-epithelial-cap (AEC)] (dark pink) associated with regeneration (Christensen and Tassava, 2000). Afterward, the AEC leads to blastema formation and subsequent outgrowth (Tassava and Loyd, 1977). Although the presence of the AEC in early and mid-stages of regeneration is demonstrated by tissue morphology and staining assessments (e.g., Han et al., 2001), its presence in late stages is observed only with tissue morphology assessment.
FIGURE 2. The wound epidermis (WE) formation is not associated with regenerative success in diverse appendage regeneration models. (A, B)
Xenopus laevis tadpoles lose their limb and tail regeneration abilities at specific developmental stages. Upon amputations, these animals cannot form an AEC (dark pink) at these stages but can still form the WE (light blue; Beck et al., 2009). (A)
X. laevis tadpole limb regeneration ability is associated with the successful specification of AER cells to form the AEC; regeneration-incompetent tadpoles cannot specify AER cells but can still seal the amputation plane, hence they can form the WE (Aztekin et al., 2021). (B)
X. laevis tail regeneration depends on the ability to relocalize their regeneration-organizing cells (ROCs; green) to amputation plane to act as the AEC (Aztekin et al., 2019). Regeneration-incompetent tadpoles cannot relocalize their ROCs, but can still seal the amputation plane, hence they can form the WE. (C) Repeated amputation of the axolotl limb results in no regeneration that can form a WE (Bryant et al., 2017b). (D) Mouse digit tip regeneration is amputation position-dependent, yet both distal regenerative and proximal regeneration deficient amputations result in the WE formation (Simkin et al., 2015).
