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Tissue and organ regeneration is a remarkable ability observed in many animal species, which has been significantly reduced or lost in several vertebrate lineages, partly due to the evolutionary loss of regeneration-associated genes. For example, many genes involved in limb and skin regeneration in anamniotes (fish and amphibians) have been lost in amniotes (reptiles, birds, and mammals) following their transition to terrestrial life. This raises the intriguing question of whether reintroducing such lost genes could partially restore regenerative abilities. Here, we investigated whether the ag1 gene, which plays a key role in regeneration in fish and amphibians and was lost in amniotes, could enhance skin wound healing in mice. We generated transgenic mice with inducible expression of the Xenopus laevis ag1 gene in the skin and compared wound healing dynamics between induced and non-induced groups. Induction of ag1 resulted in approximately 20% faster wound closure. Transcriptomic analysis revealed enhanced activation of multiple pathways involved in wound repair and, notably, the upregulation of a subset of genes typically associated with scarless healing in amphibians and mammalian fetuses. Altogether, our findings demonstrate that a gene lost more than 300 million years ago can still stimulate reparative processes in mammalian tissues, highlighting the potential of ancient gene reactivation to enhance tissue repair in modern vertebrates.
FIGURE 1. Effects of recombinant Ag1 protein on cell proliferation and migration. (A) Representative immunohistochemical staining for Ki67 in human dermal fibroblasts (hdF) cultures grown with or without recombinant Ag1. No significant differences in proliferation were observed between groups. Bar: 50 µm. (B) Kinetics of wound closure in HaCaT keratinocyte scratch assays. The graph depicts the percentage of wound healing area over time for control HaCat cells (blue) and HaCat cells treated with Ag1 (HaCaT + Ag1, red). Data are presented as the mean ± standard deviation (shaded area). The HaCaT + Ag1 group exhibited a significantly accelerated rate of wound closure compared to the untreated control.
FIGURE 2. Schematic representation of Xenopus laevis ag1 expression induction in transgenic mice.
FIGURE 3. Analysis of ag1 expression in HEK293T cells and transgenic mice. All data presented as the average of three independent replicates with standard deviations. Statistical significance was assessed by a paired Student’s t-test. (A) Real-time quantitative PCR (RT-qPCR) results for ag1 mRNA levels in HEK293T cells are depicted before and after expression of ag1-encoding lentiviral construct. (B) RT-qPCR analysis of ag1 expression in HEK293T cells co-transfected with pCre-BFP, prtTA, and pKB2-ag1 before and after doxycycline induction. (C) RT-qPCR measurement of ag1 mRNA in skin from Cre/rtTA/ag1 mice prior to and following 7 days of doxycycline administration. (D) Western blot analysis of skin lysates from Cre/rtTA/ag1 mice before and after 7 days of doxycycline treatment, probed with Ag1-specific antibodies.
FIGURE 4. Analysis of Ag1 effects in ag1 transgenic mice. (A) [NOT SHOWN] Schematic representation of the full-thickness fixed-splint skin wound model. (B) Effect of ag1 expression on wound closure dynamics in vivo in transgenic versus control mice. The x-axis represents the days after the surgery (day 1) and the y-axis represents the percentage of the original wound size remaining. * marks the significant difference between experimental and control groups (p < 0.05).
FIGURE 5 Transcriptomic impact of ag1 expression in wounded skin. Volcano plot of differentially expressed genes in ag1-transgenic versus control mice. Genes with a fold change ≥2 or ≤ −2 (|log2 fold change| ≥ 1) and padj <0.05 were considered significantly regulated. Ginger and blue dots indicate downregulated activated genes, respectively. Grey dots indicate non-significant changes.
FIGURE 6. Differential gene expression and pathway enrichment in ag1-expressing wounds. (A) Heatmap of all differentially expressed genes (DEGs), showing clear clustering of biological replicates and distinct transcriptional profiles between ag1-transgenic and control wound tissues. (B) Enrichment analysis of downregulated DEGs identifies processes related to trans-synaptic signaling, excitatory signaling, such as trans-synaptic signaling, cilium movement, and response to calcium. (C) Enrichment analysis of upregulated DEGs highlights biological processes associated with tissue repair, including extracellular matrix organization, regulation of cell migration, vascular development, collagen biosynthesis, and wound response.
FIGURE 7. Ag1 expression reactivates genes characteristic of scarless wound healing. (A) Heatmap showing overlap between upregulated genes in ag1-transgenic mice and a curated gene set associated with fetal scarless repair (see Supplementary Table S2). Hallmark regenerative genes such as Col3a1 (Collagen type III), Tgfβ3, Prrx1, Prrx2, Tnc (Tenascin-C), Twist1 and Twist2 were consistently upregulated, mirroring fetal regeneration programs. (B) Positions of the genes shown in heatmap A on the volcano plot in Figure 5.
FIGURE 5. Transcriptomic impact of ag1 expression in wounded skin. Volcano plot of differentially expressed genes in ag1-transgenic versus control mice. Genes with a fold change ≥2 or ≤ −2 (|log2 fold change| ≥ 1) and padj <0.05 were considered significantly regulated. Ginger and blue dots indicate downregulated activated genes, respectively. Grey dots indicate non-significant changes.
