A cellular, molecular, and pharmacological basis for appendage regeneration in mice

Abstract

Regenerative medicine aims to restore normal tissue architecture and function. However, the basis of tissue regeneration in mammalian solid organs remains undefined. Remarkably, mice lacking p21 fully regenerate injured ears without discernable scarring. Here we show that, in wild-type mice following tissue injury, stromal-derived factor-1 (Sdf1) is up-regulated in the wound epidermis and recruits Cxcr4-expressing leukocytes to the injury site. In p21-deficient mice, Sdf1 up-regulation and the subsequent recruitment of Cxcr4-expressing leukocytes are significantly diminished, thereby permitting scarless appendage regeneration. Lineage tracing demonstrates that this regeneration derives from fate-restricted progenitor cells. Pharmacological or genetic disruption of Sdf1-Cxcr4 signaling enhances tissue repair, including full reconstitution of tissue architecture and all cell types. Our findings identify signaling and cellular mechanisms underlying appendage regeneration in mice and suggest new therapeutic approaches for regenerative medicine.

Keywords: developmental biology; genetics; molecular biology; regenerative medicine; tissue regeneration.

Figure 1.

Restriction of ectoderm fates in the regenerating ear. (A) Photographs of wounded wild-type and p21^−/− mouse ears on days 0 and 28. Representative images were selected from each group. (B) The area of the wounded ear hole measured in wild-type and p21^−/− mice on weeks 1, 2, 3, and 4. n = 12–14 mice per group. (*) P < 0.01. The experiment was performed four times independently. (C) Schematic for histology orientation. The red square represents a sample histology section. (D) H&E staining of vertical cross-sections taken from the rim of the wound hole in wild-type and p21^−/− mice at day 28. Magnification, 2.5×. Solid vertical lines indicate the ends of the cartilage plate; broken lines demonstrate soft tissue borders. (E) H&E staining of vertical cross-sections taken from the rim of the wound hole in wild-type and p21^−/− mice at day 14. (F) Vertical cross-sections of K14CreER^mTmG appendages taken from the rim of the wound hole at baseline and 1–4 wk after injury for wild-type and p21^−/− mice. GFP fluorescence (green) marks lineage-traced keratinocytes. n = 5–6 mice per time point. For the orientation of the histology panels, the wound hole is located at the right of the section and is denoted by “hole.” Unless noted, all histologic images were obtained at 10× magnification. Nuclei are blue from DAPI counterstain. (e) Epidermis; (d) dermis; (c) cartilage; (hf) hair follicle. Data are presented as average ± SEM. Unless noted, all experiments were performed two to three times independently.

Figure 2.

Restriction of mesoderm fates in the regenerating ear. (A) Vertical cross-sections of Col2-CreER^mTmG appendages taken from the rim of the wound hole at baseline and 2–3 wk after injury for wild-type and p21^−/− mice. GFP fluorescence (green) marks lineage-traced chondrocytes. The dotted line represents the approximate plane of wounding. Red arrows denote new cartilage generation. n = 5–6 mice per time point. (B) Percentage of chondrocytes expressing Ki-67 in wild-type and p21^−/− mice at 4-wk after injury. n = 4 mice per group. (*) P < 0.01. (C) Vertical cross-sections of Tie2Cre^mTmG appendages taken from the rim of the wound hole at 2 wk after injury for wild-type and p21^−/− mice. CD31/CD45 cells were stained with Cy5 (red), and GFP fluorescence (green) marks lineage-traced cells. n = 5–6 mice per time point. For orientation of histology panels, the wound hole is located at the right of the section and is denoted by “hole.” Magnification, 10×. Nuclei are blue from DAPI counterstain. (e) Epidermis; (d) dermis; (c) cartilage. Data are presented as average ± SEM. All experiments were performed two to three times independently.

Figure 3.

Lack of Sdf1 expression in p21^−/− wounded keratinocytes. (A) Cells were isolated from the rim of a wounded ear appendage at different time points. (B) Relative mRNA levels of α-smooth muscle actin (SMA) isolated from the injury rim in wild-type and p21^−/− mouse ears at baseline and weeks 1–2. n = 4–5 mice per time point. (*) P < 0.05. (C) Trichome staining on vertical cross-sections of wild-type and p21^−/− ear appendages taken from the rim of the wound hole at 1 wk after injury. (Blue) Collagen and cartilage; (red) keratin; (black) nuclei. For orientation, the wound hole is located at the right of the section for all panels. Magnification, 10×. (D) Relative mRNA levels of Sdf1 in the injury rim of wild-type or p21^−/− mouse ears. n = 3–4 mice per time point. (*) P < 0.03. (E) Relative mRNA levels of Sdf1 in different cell types isolated from the injury rim in wild-type and p21^−/− mice at baseline and week 1. (*) P = 0.005. n = 4–5 mice per time point. (F) SDF1 immunostaining (green) on vertical cross-sections of wild-type and p21^−/− mouse ear appendages taken from the rim of the wound hole at 1 wk after injury. For orientation, the wound hole is located at the right of the section for all panels. (G, left panel) Relative mRNA levels of Cdkn1a in wild-type and Cdkn1a knockdown primary human keratinocytes. (Right panel) Relative mRNA levels of Sdf1 in baseline, nutrient-deprived, and hypoxia-treated wild-type and p21^−/− Cdkn1a knockdown keratinocytes. (*) P < 0.005. (H, left panel) Relative mRNA levels of Cebpa in wild-type and Cebpa knockdown mouse embryonic fibroblasts. (*) P < 0.001. (Right panel) Relative mRNA levels of Sdf1 in baseline, nutrient-deprived, and hypoxia-treated wild-type and Cebpa knockdown keratinocytes. (*) P < 0.005; (**) undetectable transcript levels and P < 0.005. Data are presented as average ± SEM. All experiments were performed two to three times independently.

Figure 4.

Failure of Cxcr4⁺ leukocyte recruitment to the wound rim in p21^−/− mice. (A,B) Relative mRNA levels (A) and protein levels (B) of Cxcr4 in the injury rim of wild-type and p21^−/− mouse ears at the indicated times. n = 4–5 mice per time point. (*) P < 0.01. (C) GFP⁺ cells from Tie2Cre^mTmG mice were isolated and further divided into CD45⁺ leukocyte and CD45^neg endothelial cell fractions. Relative mRNA levels of Cxcr4 were compared between wild-type and p21^−/− mice at baseline (BL) and 1 wk after injury. (D) Relative mRNA levels of Cxcr4 in endothelial cells (EC) and leukocytes (WBC). n = 6–7 samples per group. (*) P < 0.02. (E) CD45 and CXCR4 immunostaining on vertical cross-sections taken from the rim of the wound hole in wild-type and p21^−/− mouse appendages at 1 wk after injury. For orientation, the wound hole is located at the right of the section for all panels. (F) Percentage of leukocytes, T cells, and endothelial cells in the total population of sorted cells from the rim of a wounded ear in wild-type and p21^−/− mice at 1 wk after injury. n = 4–8 per treatment group. (*) P < 0.04. Data are presented as average ± SEM. All experiments were performed two to three times independently.

Figure 5.

Genetic or pharmacologic inhibition of Sdf1–Cxcr4 signaling in wild-type mice promotes tissue regeneration. (A) Photographs of control and AMD3100-treated mouse ears at week 4. (B) The area of the hole in the wounded ear at the indicated times for control and AMD3100 treatment groups. n = 8–9 for each treatment group. (*) P < 0.003. (C) Relative mRNA levels of α-smooth muscle actin (SMA) at day 4 for each treatment group. n = 3–4 for each treatment group. (*) P < 0.001. (D) Relative mRNA levels of Sdf1 and Cxcr4 at day 4 for each treatment group. n = 3–4 for each treatment group. (*) P = 0.04. (E) Trichrome staining of vertical cross-sections taken from the rim of the wound hole in control and AMD3100-treated appendages on day 14. (Blue) Collagen and cartilage; (red) keratin; (black) nuclei. The black arrow indicates new cartilage synthesis. The dotted line represents the approximate plane of wounding. Magnification, 10×. (F) The percentage of chondrocytes expressing Ki-67 for each treatment group. (*) P = 0.02. (G) The area of the hole in the wounded ear at the indicated times for control and 1-wk AMD3100 pulse treatment groups. n = 8–9 for each treatment group. (*) P < 0.01. (H, left panel) Relative mRNA levels of Cxcr4 in hearts and ears isolated from untreated and tamoxifen-treated Cxcr4^f/f Rosa26CreER^T2 mice. n = 3 for each treatment group. (Right panel) The area of the hole in the wounded ear at the indicated times in untreated and tamoxifen-treated Cxcr4^f/f Rosa26CreER^T2 mice. n = 7–8 for each treatment group. (*) P < 0.01. Data are presented as average ± SEM. Representative images were selected for each group. All experiments were performed two to three times independently.

Figure 6.

Chemokine signals from wound epidermis regulate tissue regeneration in mice. (Left panel) An illustration depicting how wounded wild-type keratinocytes induce Sdf1 to recruit Cxcr4⁺ leukocytes to promote fibrosis and scar formation. (Right panel) In wounded p21^−/− animals, Sdf1 induction is markedly diminished, and fewer Cxcr4⁺ leukocytes are recruited, thereby permitting lineage-restricted tissue regeneration. Genetic or pharmacologic disruption of Sdf1–Cxcr4 signaling enhances tissue repair.