TY - JOUR
AU - Alman, Benjamin A.
AB - Abstract During skin wound healing, fibroblast-like cells reconstitute the dermal compartment of the repaired skin filling the wound gap. A subset of these cells are transcriptionally active for β-catenin/T-cell factor (TCF) signaling during the proliferative phase of the repair process, and β-catenin levels control the size of the scar that ultimately forms by regulating the number of dermal fibroblasts. Here, we performed cell lineage studies to reveal a source of the dermal cells in which β-catenin signaling is activated during wound repair. Using a reporter mouse, we found that cells in the early wound in which TCF-dependent transcription is activated express genes involved in muscle development. Using mice in which cells express Pax7 (muscle progenitors) or Mck (differentiated myocytes) are permanently labeled, we showed that one quarter of dermal cells in the healing wound are Pax7 expressing progeny, but none are Mck progeny. Removing one allele of β-catenin in Pax7 expressing progeny resulted in a significantly smaller scar size with fewer Pax7 expressing progeny cell contributing to wound repair. During wound healing, β-catenin activation causes muscle satellite cells to adopt a fibrotic phenotype and this is a source of dermal cells in the repair process. Healing, Wound, Beta-catenin, Muscle, Satellite cells, Pax7, Mck Introduction In normal skin, the epidermis and dermis form a protective barrier against the external environment. Once the protective barrier is broken, the process of wound healing is set in motion. This is an intricate process involving multiple cell types and signaling pathways. It is divided into sequential, yet overlapping stages: hemostasis, inflammatory, proliferation, and remodeling [1]. These stages must be coordinated and timed to restore tissue integrity. Skin does not completely regenerate, but instead scar formation is the minimal consequence of a skin injury [1–3]. An insufficient or excessive healing response results in either a lack of healing or a hyperplastic scar, both conditions have major deleterious effects, and as such are a major clinical problem affecting approximately 5% of the population. The proliferative phase of healing, which is characterized by collagen deposition, granulation tissue formation, reepithelialisation, and wound contraction, plays a critical role in the future fate of healing and regulating the size of the scar that ultimately forms. A prominent role for β-catenin, a key mediator in the canonical Wnt signaling pathway, has been demonstrated during this phase of healing [2]. The canonical Wnt signaling pathway acts by regulating ubiquitin-mediated β-catenin degradation, in which a multiprotein complex, including GSK-3β plays a role [4]. Highlighting the important role of this signaling pathway, GSK-3β also has been shown to regulate scar size in the mouse [5]. Inhibition of ubiquitin-mediated β-catenin degradation, results in the accumulation and subsequent nuclear translocation of β-catenin, which induces the expression of cell type-specific target genes [6]. During the proliferative phase of wound repair, a subset of dermal cells exhibit increased β-catenin-mediated T-cell factor (TCF)–dependent transcriptional activation and this activation returns to baseline levels when the proliferative phase ends [2]. β-catenin levels regulate fibroblast proliferation during the proliferative phase of repair, mediate the effects of growth factors in wound repair, and regulate the ultimate size of the scar that forms [7]. The source of the cells in which β-catenin signaling is activated is not known. One potential source for dermal cells in which β-catenin signaling is activated during the proliferative phase of wound repair is from local mesenchymal cells, such as those derived from muscle cells. Indeed, the pace of wound repair is compromised when muscle does not underlay a full thickness skin defect, suggesting muscle as an important source of cells for wound repair [8]. In addition, β-catenin signaling cause muscle satellite cells to adopt a fibrotic phenotype, similar to the cytological phenotype of the dermal cells observed during the proliferative phase of wound repair [9]. Here, we examined the source of TCF transcriptional active cells in wound repair. Expression analysis of TCF transcriptional active cells suggested myocytes as a potential source of the cells during skin healing. Through cell lineage study, using muscle progenitor (Pax7) reporter mouse, we showed that Pax7 expressing cells contribute to wound repair, changing their cytologic phenotype in response to injury. This phenotype change is regulated by the key molecule of Wnt signaling pathway, that is, β-Catenin. Materials and Methods Mice MCK-Cre (B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J) [10] mice express Cre recombinase driven by the muscle creatine kinase (MCK or Ckm) promoter, and Pax7-Cre (Pax7-ires-Cre) express Cre recombinase driven by the Pax7 regulatory elements [11]. ROSA-EYFP (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J) [12] mice contain an enhanced yellow fluorescent protein gene inserted into the Gt(ROSA)26Sor locus. Expression of EYFP is blocked by an upstream loxP-flanked stop sequence. When bred to mice expressing a Cre recombinase in a tissue specific manner, the stop sequence of the targeted gene is deleted in the tissue of interest and EYFP is expressed. Catnbtm2Kem mice [13], which possess loxP sites flanking introns 1 and 6 of the gene encoding β-catenin, result in a null allele when treated with a Cre recombinase. TCF reporter mice [2] contain a LacZ gene downstream of a c-fos minimal promoter and three consensus TCF-binding motifs. Animal protocols and usage were approved by the local animal care committee. Wounding and Wound Analysis Approximately 10-week-old mice were anesthetized and the dorsal skin was shaved. After sanitizing with 70% ethanol, four full-thickness wounds were created on the dorsal skin using 4 mm diameter dermal biopsy punches (Miltex Inc., York, PA, www.miltex. com). Wound tissues were excised from mice at 1 week postwounding, fixed in 10% formalin overnights, and subjected to paraffin section at 5 μm thickness and subsequently stained. To measure scar width, 5 μm paraffin sections of wounds were subjected to Trichrome staining (Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich.com/customer-service.html) according to the manufacturer's manual. Using this method, collagen fibers stained blue, nuclei stained black, and cytoplasm and muscle fibers stained red. Wound size was determined using histological sections cut at a right angle to the skin surface across the wound. Serial sections were observed, and the section at the center of the wound, with the largest wound diameter, was chosen to measure wound size. The software Meazure 2.0 (C Thing Software, Mountain View, CA, www.cthing.com/Meazure.asp) was used to measure the size of mesenchymal (or dermal) component of each wound as described previously [2]. Immunohistochemistry Analysis Approximately 5 μm fixed sections were subjected to immunohistochemistry using an antibodies to GFP (Rockland Inc., Gilbertsville, PA, www.rockland-inc.com/ContactUs/Contact.aspx), Keratin 14 (Covance, Montreal, QC, Canada, https://store.crpinc.com/datasheet.aspx?catalogno=PRB-155P), Pax7 (Developmental Hybridoma Studies Bank, Iowa City, Iowa, dshb.biology.uiowa.edu), fibroblast activation protein (FAP) (Abcam, Cambridge, MA, www.abcam.com), α-smooth muscle actin (ASM) (Abcam) and/or β-galactosidase (Abcam). After washing, the samples were incubated with biotinylated secondary antibodies (Vector Laboratory, Burlington, ON, Canada, www.vectorlabs.com/canada/default. aspx), followed by incubation with DyLight conjugated Streptavidin. Sections were counter stained using 4′,6-diamidino-2-phenylindole (DAPI) (VECTASHIELD Mounting Medium with DAPI). Five high powered fields were examined at regular intervals across the wound for each wound to count the number of cells with positive and negative staining for each sample. AxioVision software (Zeiss, Thornwood, NY, www.zeiss.com) was used to assist in the cell counting. LacZ expression was detected by incubating wound samples in 5-bromo-4-chloro-3-indolyl β-D-galactoside staining solution according to a protocol obtained from Specialty Media (Lavellette, NJ, www.millipore.com/stemcell/stm4/custom_media_services) [2]. Flow Cytometry The wound was dissected from the unwounded tissue under microscopy 7 days postwounding. For flow cytometry of β-Galactosidase positive cells, after washing, the dissociated cells fixed and permeablized, using BD Cytofix/Cyto perm buffer by incubating them for 20 minutes at room temperature. After washing, the cells resuspended in staining buffer containing fluorescein di-β-D-galactopyranoside (FDG) substrate, which gives a green fluorescent product in the β-Gal positive cells. Next, cells stained with 1 μg/ml of propidium iodide (Molecular probe, Eugene, OR, www.invitrogen.com/site/us/en/home/brands/Molecular-Probes.html). Nonviable cells were excluded from analysis. MoFlow (Cytomation, Miami, FL, www.coulterflow.com/bciflow/instruments02.php) using a blue laser (488 nm) power set to 100 mW using FL1 with 530/40BP filter to detect EYFP expressing cells. Gene Expression Study of Wound Tissues Microdissected wound skin tissue was homogenized using a metal tissue smasher which precooled in liquid nitrogen. Total RNA was extracted using Qiagen kit according to manufacturer's manual. RNA (1 μg) was used for reverse transcription using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, www.invitrogen. com/site/us/en/home/support.html). The cDNA was subjected to real-time polymerase chain reaction (PCR) as described before [14] to detect mRNA level. All reactions were run on an Applied Biosystems 7900 HT Fast real-time PCR machine. For microarray, RNA was extracted as mentioned and the quality of RNA was assessed with a Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada, www.home.agilent.com/agilent/home.jspx?cc=CA&lc=eng). cDNA was generated and hybridized onto the Affymetrix Mouse Gene 1.0 ST chips. To compare β-Gal+ cells expression data with β-Gal-cells, an analysis of gene expression was performed using Parktec Genotyping Suite and Ingenuity Systems Software. Statistics The data are presented as mean ± 95% confidence interval unless specified. Two-tailed Student's t test was used for analysis. A p value less than 0.05 was considered statistically significant. Results A Subset of β-Catenin-Mediated TCF-Dependent Cells in Wound Repair May Drive from Muscle To determine the source of cells in which β-catenin-mediated TCF-dependent signaling is activated, we examined wound repair using a LacZ TCF reporter mouse [2]. Cells were dissociated from the dermal component of the 1-week-old wounds and sorted using FDG [15]. LacZ expressing cells were sorted from the reminder of cells and gene expression analyzed using Affymetrix mouse 1.0 ST gene array. Comparison of expression arrays (Gene Expression Omnibus database, accession no. GSE30913) using ingenuity pathway analysis [16] showed activation of skeletal muscular system development and function as one of the top functional pathways activated in LacZ positive cells when compare with LacZ negative cells (Supporting Information Table S1). Real-time PCR was used to verify the gene expression changes. In addition, using the TCF-reporter mouse, we observed an enrichment of cells exhibiting β-catenin-mediated TCF-dependent transcriptional activity adjacent to the muscle layer underlying the skin [17] during the early phases of wound repair, further supporting the notion of muscle as one of the source of this cell population (Fig. 1). 1 Open in new tabDownload slide A subpopulation of cells in the healing wound is transcriptionally active for β-catenin/TCF. (A, B): β-catenin transcriptionally active cells are responsible for about one-third of cells in the healing wound. Cells isolated from the healing wounds were analyzed using flow cytometry for the proportion of cells expressing β-Galactosidase. Roughly, one-third of cells express β-Galactosidase. Numbers represent the percentage of cells within area gated for TCF-activity. (A) is unstained sample and (B) is stained sample. (C–E): The proportion of β-Galactosidase positive cells increases during the proliferative phase of wound repair. (C): For 3 days, (D) for 1 week, and (E) for 2 weeks postwounding. (F): The proportion of β-Galactosidase positive cells at different time point of postwounding. Data is given as the mean and 95% confidence interval of percentage of β-Galactosidase positive cells. Abbreviations: FDG, fluorescein di-D-galactopyranoside; FITC, fluorescein isothiocyanate. 1 Open in new tabDownload slide A subpopulation of cells in the healing wound is transcriptionally active for β-catenin/TCF. (A, B): β-catenin transcriptionally active cells are responsible for about one-third of cells in the healing wound. Cells isolated from the healing wounds were analyzed using flow cytometry for the proportion of cells expressing β-Galactosidase. Roughly, one-third of cells express β-Galactosidase. Numbers represent the percentage of cells within area gated for TCF-activity. (A) is unstained sample and (B) is stained sample. (C–E): The proportion of β-Galactosidase positive cells increases during the proliferative phase of wound repair. (C): For 3 days, (D) for 1 week, and (E) for 2 weeks postwounding. (F): The proportion of β-Galactosidase positive cells at different time point of postwounding. Data is given as the mean and 95% confidence interval of percentage of β-Galactosidase positive cells. Abbreviations: FDG, fluorescein di-D-galactopyranoside; FITC, fluorescein isothiocyanate. Pax7 Expressing Cells (Satellite Cells) but Not Mature Myocytes Contribute to the Dermal Component of Wound Repair As β-catenin regulates dermal fibroblasts during skin healing [7] and plays a role in the conversion of muscle cells to a fibrogenic phenotype [9], it is possible that the fibrous cells present in dermal component of scar originates from muscle cells or their progenitors. To test for this possibility, we examined skin wound repair in mice in which we could label either Pax7 expressing cells or terminally differentiated skeletal muscle cells. Satellite cells, which are characterized by the expression of Pax7 [18], are mitotically quiescent in adult muscle; they can be activated when needed to generate myoblasts, which eventually differentiate to provide new myonuclei for the homeostasis, hypertrophy, and repair of muscle fibers, or fuse together to form new myofibres for regeneration [19]. Satellite cells also self-renew to maintain a viable stem-cell pool that is able to respond to repeated demand. Beside satellite cells, pericytes are another source of muscle progenitor cells, but these cells do not express markers of satellite cells, in particular they do not express Pax7 [20]. Mice expressing Cre recombinase, driven by the regulatory elements of either Mck (muscle creatine kinase for skeletal muscle) or Pax7 (satellite cells) were crossed with mice containing the Gt(ROSA)26Sor-EYFP sequence allele to generate mice in which Pax7 expressing cells or differentiated skeletal muscle cells would express EYFP (Fig. 2A). 2 Open in new tabDownload slide Pax7 expressing progeny contribute to the dermal compartment of wound repair. (A): Schematic of mouse models used in the study. Pax7-Cre and Mck-Cre transgenic mice were crossed with Gt(ROSA)26Sortm1(EYFP)Cos/J mice to label cells expressing pax7 (satellite cellls) or Mck (differentiated myocytes) to determine the proportion of the various cell types that contribute to wound repair. Pax7-Cre and Mck-Cre transgenic mice were crossed with Catnbtm2Kem(fl/fl). β-Catenin is deleted when cre-recombinase is expressed in mice expressing the Catnbtm2Kem(fl/fl) allele. These mice were then bred with EYFP reporter mouse (Gt(ROSA)26Sortm1(EYFP)Cos/J) to identify the cells in which recombination occurred. To monitor β-Catenin/TCF transcriptional activity, the reporter mice were bred with the TCF reporter mouse. TCF transcriptional activity is identified in cells by the production of β-Galactosidase. (B): Skeletal muscle cells from Pax7-Cre; Rosa.EYFP mice which injured through full thickness biopsy are positive for EYFP, showing that satellite cells and their progeny (mature myocytes) are labeled in these mice. EYFP+ cells (Pax7 progeny) are observed throughout repaired wound up to keratinocyte layer. Arrowhead shows EYFP+ fibroblast-like cells underneath the keratinocyte layer and arrows represent the muscle layers both sides of healed skin. Blue staining is DAPI, which labels the nuclei. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TCF, T-cell factor. 2 Open in new tabDownload slide Pax7 expressing progeny contribute to the dermal compartment of wound repair. (A): Schematic of mouse models used in the study. Pax7-Cre and Mck-Cre transgenic mice were crossed with Gt(ROSA)26Sortm1(EYFP)Cos/J mice to label cells expressing pax7 (satellite cellls) or Mck (differentiated myocytes) to determine the proportion of the various cell types that contribute to wound repair. Pax7-Cre and Mck-Cre transgenic mice were crossed with Catnbtm2Kem(fl/fl). β-Catenin is deleted when cre-recombinase is expressed in mice expressing the Catnbtm2Kem(fl/fl) allele. These mice were then bred with EYFP reporter mouse (Gt(ROSA)26Sortm1(EYFP)Cos/J) to identify the cells in which recombination occurred. To monitor β-Catenin/TCF transcriptional activity, the reporter mice were bred with the TCF reporter mouse. TCF transcriptional activity is identified in cells by the production of β-Galactosidase. (B): Skeletal muscle cells from Pax7-Cre; Rosa.EYFP mice which injured through full thickness biopsy are positive for EYFP, showing that satellite cells and their progeny (mature myocytes) are labeled in these mice. EYFP+ cells (Pax7 progeny) are observed throughout repaired wound up to keratinocyte layer. Arrowhead shows EYFP+ fibroblast-like cells underneath the keratinocyte layer and arrows represent the muscle layers both sides of healed skin. Blue staining is DAPI, which labels the nuclei. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TCF, T-cell factor. We first examined the native skin of the mice and found that there was no expression of EYFP in dermal layer of unwounded skin in mice in which EYFP expression was driven by either Pax7 or Mck, but the myocytes and satellite cells were labeled as expected. Importantly, EYFP positive cells were not observed in any other cell types in the mice, including vascular cells and cells surrounding blood vessels (Fig. 3A, 3B). We then examined wound repair by producing 4 mm diameter full thickness wounds in the animals. One week after wounding, fluorescent microscopy revealed that in Pax7-Cre; ROSA-EYFP mice, EYFP expressing cells were present in the dermal layer of the wound (Figs. 2B, 3D). We also examined the expression of Pax7 during wound repair at 3, 5, and 7 days postwounding and did not identify the expression of this gene by either RNA or protein analysis during the repair process (Supporting Information Figs. S1, S2). 3 Open in new tabDownload slide Pax7 expressing progeny but not Mck expressing progeny are present in dermal component of healing skin. (A): Skeletal muscle cells from MCK-Cre; Rosa.EYFP mice express EYFP, showing that mature muscle cells are labeled in this mouse. (B): Skeletal muscle cells from Pax7-Cre; Rosa.EYFP mice are positive for EYFP, showing that satellite cells and their progeny (mature myocytes) are labeled in these mice. (C): The central portion of the wound 1 week after wound generation in MCK-Cre; Rosa.EYFP mice. No EYFP+ cells are seen in the wound indicating that mature myocytes do not contribute to wound repair. Dashed line is highlighting the dermal-epithelial junction. (D): The central portion of the wound 1 week after wound generation in Pax7-Cre; Rosa.EYFP mice. Arrowhead shows EYFP+ fibroblast-like cells underneath the keratinocyte layer, showing that Pax7 expressing cells (satellite cells) or their progeny contribute to wound repair. Dashed line is highlighting the dermal-epithelial junction. Blue staining is DAPI, which labels the nuclei. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Mu, muscle cells. 3 Open in new tabDownload slide Pax7 expressing progeny but not Mck expressing progeny are present in dermal component of healing skin. (A): Skeletal muscle cells from MCK-Cre; Rosa.EYFP mice express EYFP, showing that mature muscle cells are labeled in this mouse. (B): Skeletal muscle cells from Pax7-Cre; Rosa.EYFP mice are positive for EYFP, showing that satellite cells and their progeny (mature myocytes) are labeled in these mice. (C): The central portion of the wound 1 week after wound generation in MCK-Cre; Rosa.EYFP mice. No EYFP+ cells are seen in the wound indicating that mature myocytes do not contribute to wound repair. Dashed line is highlighting the dermal-epithelial junction. (D): The central portion of the wound 1 week after wound generation in Pax7-Cre; Rosa.EYFP mice. Arrowhead shows EYFP+ fibroblast-like cells underneath the keratinocyte layer, showing that Pax7 expressing cells (satellite cells) or their progeny contribute to wound repair. Dashed line is highlighting the dermal-epithelial junction. Blue staining is DAPI, which labels the nuclei. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Mu, muscle cells. To detect the phenotype of the Pax7 progeny cells, we double stained the EYFP expressing cells with either FAP, as a fibroblast marker [21] or with ASM as the marker of myofibroblast [22]. Although the majority of EYFP+ cells in dermal component of healing wound stained for FAP (73% ± 9) but a subpopulation (14% ± 8) of EYFP+ cells stained for ASM mainly at the junction of healing dermis with intact skin (Supporting Information Fig. S3). To evaluate the long-term phenotype of Pax7 progeny in healing skin, we harvested the wounded skin at 6 weeks postwounding and stained for EYFP. We observed EYFP+ cells at this time point in the healing dermis (Supporting Information Fig. S4). Unlike mice expressing Cre driven by Pax7, the MCK-Cre; ROSA.EYFP mice did not show any EYFP expressing cells in the wound (Fig. 3C). A Subset of Pax7 Progeny Exhibit β-Catenin/TCF Transcriptional Activity During Skin Wound Repair To determine if the Pax7 progeny are also the cells in which β-Catenin-mediated TCF transcription is active, we crossed the mice in which Pax7 drove expression of EYFP (Pax7-Cre; ROSA-EYFP) with our TCF reporter mice [2]. The triple transgenic mice express EYFP in Pax7 progeny and LacZ in cells in which β-Catenin-mediated TCF transcription is activated (Fig. 2A). Using the Pax7-Cre; ROSA.EYFP; TCF reporter mice (in which we could monitor β-catenin/TCF transcriptional activity in Pax7 progeny), we examined the proportion of EYFP+ cells that also exhibited β-Catenin/TCF transcriptional activity. While 27% of cells in dermal compartment of the wound were EYFP+ (Fig. 4D, 4F), roughly half of these cells also exhibited β-Galactosidase activity (Fig. 4D, 4F). TCF transcriptional active cells (β-Galactosidase+ cells) were more populated near the muscle layer (Fig. 4E). 4 Open in new tabDownload slide Pax7 progeny are transcriptionally active for β-Catenin in the healing wound. One-week-old wounds from Pax7.Cre+; ROSA.EYFP; TCF mouse. Double staining of healing wound with β–Galactosidase and EYFP. Cells in which β–Galactosidase is expressed exhibit TCF transcriptional activation and EYFP labels the progeny of Pax7 expressing cells. (A): DAPI staining (B): β-Galactosidase staining visualized by single filter (red) using anti-β-Galactosidase antibody (C) EYFP staining visualized by single filter (green) (D) Merged image. Half of the EYFP+ cells in healing wound also stain for β -Galactosidase. Arrow shows cells which are positive for EYFP (green) and β-Galactosidase (red), arrowhead indicates cells that are only positive for EYFP. (E): Transcriptionally, TCF active cells are enriched near injured muscle. In TCF reporter mouse, transcriptionally active TCF cells turn into blue in the presence of substrate (in 5-bromo-4-chloro-3-indolyl β-D-galactoside). Arrow shows β-Galactosidase positive cells (blue color). (F): Quantification of TCF transcriptionally active cells in EYFP+ cells. Approximately 27% of cells in the dermal compartment of the healing wound express EYFP (Pax7 progeny), and 46% of these cells also express β-Galactosidase. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; mu, muscle layer. 4 Open in new tabDownload slide Pax7 progeny are transcriptionally active for β-Catenin in the healing wound. One-week-old wounds from Pax7.Cre+; ROSA.EYFP; TCF mouse. Double staining of healing wound with β–Galactosidase and EYFP. Cells in which β–Galactosidase is expressed exhibit TCF transcriptional activation and EYFP labels the progeny of Pax7 expressing cells. (A): DAPI staining (B): β-Galactosidase staining visualized by single filter (red) using anti-β-Galactosidase antibody (C) EYFP staining visualized by single filter (green) (D) Merged image. Half of the EYFP+ cells in healing wound also stain for β -Galactosidase. Arrow shows cells which are positive for EYFP (green) and β-Galactosidase (red), arrowhead indicates cells that are only positive for EYFP. (E): Transcriptionally, TCF active cells are enriched near injured muscle. In TCF reporter mouse, transcriptionally active TCF cells turn into blue in the presence of substrate (in 5-bromo-4-chloro-3-indolyl β-D-galactoside). Arrow shows β-Galactosidase positive cells (blue color). (F): Quantification of TCF transcriptionally active cells in EYFP+ cells. Approximately 27% of cells in the dermal compartment of the healing wound express EYFP (Pax7 progeny), and 46% of these cells also express β-Galactosidase. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; mu, muscle layer. β-Catenin Modulation in Pax7 Progeny Regulates Scar Size To evaluate the effect of β-Catenin inactivation on wound repair, we bred Pax7-Cre; ROSA.EYFP mice with mice expressing the Catnbtm2Kem(fl/fl) allele to knockdown the level of β-Catenin in the cells expressing Pax7 and their progeny. Mice homozygous for the Catnbtm2Kem allele were not viable when crossed with mice expressing Cre-recombinase driven by Pax7, and thus we used heterozygous mice to knock down β-Catenin levels. The recombined cells also expressed EYFP, and 90% of muscle fibers were EYFP+. β-Catenin levels in Pax7-Cre; Catnbtm2Kem(WT/fl); ROSA.EYFP mice were half that as seen in wild-type mice (Pax7-Cre; Catnbtm2Kem(WT/WT); ROSA.EYFP) illustrating successful recombination and inactivation of one allele of β-Catenin. Pax7-Cre; ROSA.EYFP; Catnbtm2Kem(WT/fl) mice formed significantly smaller scars (n = 7, p < .001) with fewer dermal fibroblast-like cells when compare with their wild-type littermates (Fig. 5J–5L). Moreover, Pax7-Cre; Catnbtm2Kem(WT/fl); ROSA.EYFP showed fewer EYFP+ cells in their dermal compartment of the wound (14% vs. 24%) when compared with their wild-type littermates (Fig. 5A–5I). In contrast, the number of Pax7 expressing cells in muscle of Pax7-Cre; Catnbtm2Kem(WT/fl); ROSA.EYFP mice is comparable with their wild-type littermate, showing that this difference is not due to a difference in the number of Pax7 expressing cells present in the mice. 5 Open in new tabDownload slide β-Catenin level in Pax7 progeny cells regulates scar size. (A, D, G): ROSA.EYFP mice in which Cre recombinase is not expressed do not demonstrate EYFP+ cell, with 0% gated EYFP+ cells. (A): double stained with EYFP and Keratin14 showing keratinocyte layer. (D): double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (G): Flow cytometry of healing tissue is showing 0% gated EYFP+ cells. (B, E, H): Healing wound in a pax7 reporter mouse (Pax7-Cre; ROSA.EYFP) shows one-fourth of cells in healing wound are Pax7 cells progeny. These cells are found exclusively in dermal layer (E) and not in the keratinocyte layer (B). Arrowhead indicates the EYFP+ cells. (B): double stained with EYFP and Keratin14 showing keratinocyte layer. (E): Double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (H): Flow cytometry of healing wound cells in Pax7-Cre; ROSA.EYFP mouse, showing that 23.6% of cells in the healing wound are positive for EYFP. (C, F, I): Healing wound in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mice which lacks one allele for β-catenin. There are fewer cells in the healing wound that are Pax7 progeny cells. Arrowhead indicates the EYFP+ cells. (C) Double stained with EYFP and Keratin14 showing keratinocyte layer. (F) Double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (I) Flow cytometry of wound cells in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mouse, showing fewer cells in the healing wound that are EYFP+, the Pax7 progeny. (J, K): Trichrome staining of healing wounds in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mouse (K) and its wild-type (for β-catenin) littermate (J: Catnbtm2Kem(WT/WT); ROSA.EYFP) showing a smaller scar diameter in mice deficient in β-catenin as quantified and illustrated in (L) (***, p < .001). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole. 5 Open in new tabDownload slide β-Catenin level in Pax7 progeny cells regulates scar size. (A, D, G): ROSA.EYFP mice in which Cre recombinase is not expressed do not demonstrate EYFP+ cell, with 0% gated EYFP+ cells. (A): double stained with EYFP and Keratin14 showing keratinocyte layer. (D): double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (G): Flow cytometry of healing tissue is showing 0% gated EYFP+ cells. (B, E, H): Healing wound in a pax7 reporter mouse (Pax7-Cre; ROSA.EYFP) shows one-fourth of cells in healing wound are Pax7 cells progeny. These cells are found exclusively in dermal layer (E) and not in the keratinocyte layer (B). Arrowhead indicates the EYFP+ cells. (B): double stained with EYFP and Keratin14 showing keratinocyte layer. (E): Double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (H): Flow cytometry of healing wound cells in Pax7-Cre; ROSA.EYFP mouse, showing that 23.6% of cells in the healing wound are positive for EYFP. (C, F, I): Healing wound in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mice which lacks one allele for β-catenin. There are fewer cells in the healing wound that are Pax7 progeny cells. Arrowhead indicates the EYFP+ cells. (C) Double stained with EYFP and Keratin14 showing keratinocyte layer. (F) Double stained with EYFP and Keratin14 showing dermal compartment of healing skin. (I) Flow cytometry of wound cells in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mouse, showing fewer cells in the healing wound that are EYFP+, the Pax7 progeny. (J, K): Trichrome staining of healing wounds in Pax7-Cre; Catnbtm2Kem(wt/fl); ROSA.EYFP mouse (K) and its wild-type (for β-catenin) littermate (J: Catnbtm2Kem(WT/WT); ROSA.EYFP) showing a smaller scar diameter in mice deficient in β-catenin as quantified and illustrated in (L) (***, p < .001). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole. β-Catenin Modulation in Mature Myocytes of Skin Does Not Alter Wound Phenotype To determine if β-Catenin modulation in mature myocyte alters the wound phenotype, we generated mice in which conditional β-Catenin alleles were deleted by Cre-recombinase expression, driven by the regulatory elements of Mck (muscle creatine kinase for skeletal muscle). We examined the wound phenotype in these mice and observed no significant difference in scar size of the generated wounds as well as no change in other phenotypic feature of wound repair (Fig. 6). This finding supports the concept that satellite cells (Pax7 expressing cells), but not mature myocytes regulated wound size and fibroblasts during healing as modulation of the levels of β-Catenin in cells expressing MCK-Cre (MCK-Cre; Catnbtm2Kem(fl/fl); ROSA. EYFP) did not alter wound phenotype (Fig. 6). 6 Open in new tabDownload slide β-Catenin modulation in Mck expressing cells (differentiated skeletal muscles) does not affect wound phenotype. Trichrome staining of wounds harvested 1 week postwounding reveals no significant difference in wound width (Scar) in MCK-Cre; Catnbtm2Kem(fl/fl) mice, which are null for β-Catenin in MCK+ cells, when compared with WT littermates mice (n = 7, p = .074). (A, C) MCK-Cre; Catnbtm2Kem(fl/fl) mice, (B, D)Catnbtm2Kem(fl/fl) mice, black arrow shows border of wound with intact skin and white arrow shows muscle layer in skin. 6 Open in new tabDownload slide β-Catenin modulation in Mck expressing cells (differentiated skeletal muscles) does not affect wound phenotype. Trichrome staining of wounds harvested 1 week postwounding reveals no significant difference in wound width (Scar) in MCK-Cre; Catnbtm2Kem(fl/fl) mice, which are null for β-Catenin in MCK+ cells, when compared with WT littermates mice (n = 7, p = .074). (A, C) MCK-Cre; Catnbtm2Kem(fl/fl) mice, (B, D)Catnbtm2Kem(fl/fl) mice, black arrow shows border of wound with intact skin and white arrow shows muscle layer in skin. Discussion Here, we undertook cell lineage studies to identify a source of dermal mesenchymal cells in wound healing. Our studies showed that one quarter of the cells in the healing wound are derived from Pax7 expressing progeny, but not MCK expressing progeny, suggesting skeletal muscle progenitor cells, but not differentiated myocytes, as an important source of cells in wound repair. The cells derived from Pax7 progeny were located throughout the scar (but not in keratinocyte layer) of the healed skin, showing a contribution to the dermal compartment, but not the keratinocyte component of the repair process. As we did not identify the expression of Pax7 by RNA or protein analysis during the repair process, this makes it unlikely that new cells in the healing wound transiently express Pax7 and consequently recombination generates EYFP. Our data also supports the concept that Pax7 expressing Satellite cells change their cytologic phenotype to fibroblast-like cells when β-Catenin is activated, and these cells move into the wound producing the fibroblast-like EYFP+ cells as we observed. Before this work, although little was known about the origin of the cells which contribute to dermal compartment during cutaneous wound repair [1, 23, 24], several earlier studies show that the keratinocytes in the healed skin derive from progenitor cells in multiple regions including the upper region of the hair follicle [25, 26], thus highlighting the different cellular sources of the dermal and keratinocyte compartments in skin repair. As one quarter of the cells in dermal compartments of healed skin are Pax7 expressing progeny, other cell sources also contribute to the dermal compartment of wound repair. It has been shown that the upper permanent region of the hair follicle below the sebaceous glands (commonly referred to as the bulge) contains multipotent progenitor cells which contribute to re-epithelialization [25] .This is not exclusive to bulge as the presence of multipotent stem cells outside the bulge has been shown [25–29]. Moreover, it has been shown that circulating fibrocytes contribute to the myofibroblast population in the dermal compartment of wounded skin and that they originate from the bone marrow [30]. Recent studies also show a significant role for pericytes as a potent stem cell population in the skin which contribute to healing [31]. Nevertheless, our finding that a subset of Pax7 expressing muscle progenitor cells contribute to repair explains the clinical observation that wound repair is compromised when muscle does not underlay a full thickness skin defect [8]. Our observation that an enrichment of cells expressing β-Galactosidase is located close to the muscle layer of healing skin, highlights a role for β-Catenin/TCF signaling for contribution of this Pax7 expressing progeny, and suggests that β-Catenin plays an important role in cells of the Pax7 lineage as regulators of scar size. The fact that activation of β-Catenin/TCF signaling is not exclusive to Pax7 progeny highlights other studies that show activation β-Catenin/TCF signaling in other cells types such as circulating cells and local fibroblasts [32]. The observed smaller scar in mice in which these cells lack one allele of β-Catenin could be due to β-Catenin causing a difference in behavior of the Pax7 progeny or due to β-Catenin regulating the number of Pax7 expressing progenitor cells. Wnt signaling is known to positively regulate the number of dermomyotomal progenitors during axial myogenesis [33, 34]. However, in our study, the number of Pax7 expressing cells in muscle of Pax7-Cre; Catnbtm2Kem(WT/fl); ROSA.EYFP is comparable with their wild-type littermates, suggesting that smaller scar size is not due to change in the number of Pax7 expressing progenitor cells. This finding is supported by the work of Hutcheson et al. [35] in which it was found that the number of Pax7 expressing cells in muscle of Pax7iCre/+;β-cateninD/fl2-6;R26RYFP/+ mice did not detectably change. Thus, it is likely that β-Catenin regulates the behavior of the Pax7 progeny by allowing them to attain a fibroblastic phenotype during skin wound repair in a way that inactivation of β-Catenin leads to less fibroblast-like cells and consequently smaller scar size. It is possible that β-Catenin is regulating this conversion at first step and help to provide a source of cells during healing but hyperactivation of β-Catenin in new formed (myo)fibroblasts lead to higher proliferation rate and bigger scar [7]. Therefore, in this scenario, inactivation of one allele of β-Catenin led to smaller scar. β-Catenin induces a fibrogenic fate in satellite cells, converts satellite cells from a myogenic to a fibrogenic lineage [9]. Furthermore, it has been shown that Wnt proteins have a direct role in mammalian aging, a process which is associated with fibrosis [36]. In contrast to induction of a fibrogenic lineage, Wnt signaling may promote myogenic lineage progression during development [37] and Wnts positively regulate the number of dermomyotomal muscle progenitors during axial myogenesis [33, 34]. These pleiotropies might be due to difference in timing of Wnt/β-Catenin signaling activation in different cellular contexts. Besides the canonical Wnt pathway, it is possible that some noncanonical Wnt pathways are involving in this regulation as it has been shown in other mesenchymal cells [38]. The two most well-studied pathways in noncanonical Wnt pathways are the planar cell polarity (PCP) and Wnt/calcium pathways. PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, suggesting a role for this pathway during the skin healing as the cellular movement is a crucial step in proliferation and remodelling phase of skin wound repair. Significant roles for PCP pathway [39] and Wnt/calcium pathways [40] have been shown during wound repair, raising the possibility that these noncanonical pathways may change the microenvironment of healing area, contributing to fibrous tissue formation during dermis healing. Our data shows that only Pax7 expressing satellite cells, but not mature muscle cells contribute to wound repair. These cells exist in dermal compartment up to 6 weeks postwounding. Pax7-positive cells also play a role in the regeneration of axolotl spinal cord, indicating that they may have the capacity to act as progenitors for repair in a variety of tissue types [41]. Expression of Mck is restricted to terminally differentiated striated muscle [42, 43]. Our wound healing studies on mice expressing Cre driven by the MCK regulatory elements [10] revealed no contribution of this cell type to wound repair. Moreover, inactivation of β-Catenin within MCK lineage did not change the wound phenotype. Conclusion Here, we showed in vivo, that a pool of progenitor populations can change their cytologic phenotype in response to injury. This phenotype is regulated by β-Catenin, a key molecule in the Wnt signaling pathway. It is likely that β-Catenin/TCF signaling activated by growth factors and Wnt proteins during wound repair. This activation stimulates Pax7 expressing cells to adopt a fibroblastic phenotype which then can migrate to the healing wound, contributing roughly a quarter of the dermal cells during wound repair in mouse. Although skin wound repair in mouse has its own unique features but there are several similar signaling pathways activation between human and mouse during skin wound repair. Related to our study, activation of β-Catenin-mediated transcription has been shown in fibroproliferative lesions [44] of human. Similarly, it has been shown that β-Catenin stabilization in mouse lead to a bigger scar size [7], mimicking the fibroproliferative lesions in human. The contribution of Pax7 expressing cells and their progeny during mouse skin wound healing identifies a source of cells that have the potential to be used to modulate the outcome in human deficient wound repair. Acknowledgements We thank Puviindran Nadesan, Jalil Hakimi, Huimin Wang, Michelle Tseng, and Sherry Zhao for their technical assistance and Dr Sevan Hopyan for critical reading of the manuscript. This work was supported by the Canadian Institutes of Health Research grant FRN 62788; Ontario Institute for Cancer Research (to S.A.-N.); and Canada Research Chairs Programme (to B.A.A.). Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Gurtner GC , Werner S, Barrandon Y et al. Wound repair and regeneration . Nature 2008 ; 453 : 314 – 321 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Cheon SS , Cheah AY, Turley S et al. beta-Catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds . Proc Natl Acad Sci USA 2002 ; 99 : 6973 – 6978 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Amini-Nik S , Kraemer D, Cowan ML et al. Ultrafast mid-IR laser scalpel: protein signals of the fundamental limits to minimally invasive surgery . PLoS One 2010 ; 5 : e13053 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Harada N , Tamai Y, Ishikawa T et al. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene . EMBO J 1999 ; 18 : 5931 – 5942 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Kapoor M , Liu S, Shi-wen X, et al. GSK-3beta in mouse fibroblasts controls wound healing and fibrosis through an endothelin-1-dependent mechanism . J Clin Invest 2008 ; 118 : 3279 – 3290 . Google Scholar Crossref Search ADS PubMed WorldCat 6 van Amerongen R , Nusse R. Towards an integrated view of Wnt signaling in development . Development 2009 ; 136 : 3205 – 3214 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Cheon SS , Wei Q, Gurung A et al. Beta-catenin regulates wound size and mediates the effect of TGF-beta in cutaneous healing . FASEB J 2006 ; 20 : 692 – 701 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Fischer JE , Bland KI. Mastery of Surgery . Philadelphia, PA : Wolters Kluwer/Lippincott Williams & Wilkins , 2007 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 9 Brack AS , Conboy MJ, Roy S et al. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis . Science 2007 ; 317 : 807 – 810 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Bruning JC , Michael MD, Winnay JN et al. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance . Mol Cell 1998 ; 2 : 559 – 569 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Nishijo K , Hosoyama T, Bjornson CR et al. Biomarker system for studying muscle, stem cells, and cancer in vivo . FASEB J 2009 ; 23 : 2681 – 2690 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Srinivas S , Watanabe T, Lin CS et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus . BMC Dev Biol 2001 ; 1 : 4 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Brault V , Moore R, Kutsch S et al. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development . Development 2001 ; 128 : 1253 – 1264 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Amini Nik S , Ebrahim RP, Van Dam K et al. TGF-beta modulates beta-Catenin stability and signaling in mesenchymal proliferations . Exp Cell Res 2007 ; 313 : 2887 – 2895 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Nolan GP , Fiering S, Nicolas JF et al. Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ . Proc Natl Acad Sci USA 1988 ; 85 : 2603 – 2607 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Chari R , Lonergan KM, Pikor LA et al. A sequence-based approach to identify reference genes for gene expression analysis . BMC Med Genomics 3 : 32 . Crossref Search ADS PubMed WorldCat 17 Hedrich HJ , Bullock GR. The Laboratory Mouse. The Handbook of Experimental Animals . Amsterdam, Boston : Elsevier Academic Press ; 2004 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 18 Relaix F , Montarras D, Zaffran S et al. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells . J Cell Biol 2006 ; 172 : 91 – 102 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Zammit PS . All muscle satellite cells are equal, but are some more equal than others? J Cell Sci 2008 ; 121 : 2975 – 2982 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Dellavalle A , Sampaolesi M, Tonlorenzi R et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells . Nat Cell Biol 2007 ; 9 : 255 – 267 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Rettig WJ , Garin-Chesa P, Beresford HR et al. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells . Proc Natl Acad Sci USA 1988 ; 85 : 3110 – 3114 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Jeon ES , Moon HJ, Lee MJ et al. Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells . Stem Cells 2008 ; 26 : 789 – 797 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Martin P . Wound healing—Aiming for perfect skin regeneration . Science 1997 ; 276 : 75 – 81 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Werner S , Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing . J Invest Dermatol 2007 ; 127 : 998 – 1008 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Ito M , Liu Y, Yang Z et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis . Nat Med 2005 ; 11 : 1351 – 1354 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Taylor G , Lehrer MS, Jensen PJ et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis . Cell 2000 ; 102 : 451 – 461 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Claudinot S , Nicolas M, Oshima H et al. Long-term renewal of hair follicles from clonogenic multipotent stem cells . Proc Natl Acad Sci USA 2005 ; 102 : 14677 – 14682 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Oshima H , Rochat A, Kedzia C et al. Morphogenesis and renewal of hair follicles from adult multipotent stem cells . Cell 2001 ; 104 : 233 – 245 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Rochat A , Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis . Cell 1994 ; 76 : 1063 – 1073 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Mori L , Bellini A, Stacey MA et al. Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow . Exp Cell Res 2005 ; 304 : 81 – 90 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Paquet-Fifield S , Schluter H, Li A et al. A role for pericytes as microenvironmental regulators of human skin tissue regeneration . J Clin Invest 2009 ; 119 : 2795 – 2806 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 32 Oguma K , Oshima H, Aoki M et al. Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells . EMBO J 2008 ; 27 : 1671 – 1681 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Galli LM , Willert K, Nusse R et al. A proliferative role for Wnt-3a in chick somites . Dev Biol 2004 ; 269 : 489 – 504 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Schmidt C , Otto A, Luke G et al. Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo . Anat Embryol (Berl) 2006 ; 211 : 525 – 534 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Hutcheson DA , Zhao J, Merrell A et al. Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin . Genes Dev 2009 ; 23 : 997 – 1013 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Liu H , Fergusson MM, Castilho RM et al. Augmented Wnt signaling in a mammalian model of accelerated aging . Science 2007 ; 317 : 803 – 806 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Holowacz T , Zeng L, Lassar AB. Asymmetric localization of numb in the chick somite and the influence of myogenic signals . Dev Dyn 2006 ; 235 : 633 – 645 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Arnsdorf EJ , Tummala P, Jacobs CR. Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate . PLoS One 2009 ; 4 : e5388 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Caddy J , Wilanowski T, Darido C et al. Epidermal wound repair is regulated by the planar cell polarity signaling pathway . Dev Cell 2010 ; 19 : 138 – 147 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Wang Q , Symes AJ, Kane CA et al. A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer . PLoS One 2010 ; 5 : e10456 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Schnapp E , Kragl M, Rubin L et al. Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration . Development 2005 ; 132 : 3243 – 3253 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Donoviel DB , Shield MA, Buskin JN et al. Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice . Mol Cell Biol 1996 ; 16 : 1649 – 1658 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Shield MA , Haugen HS, Clegg CH et al. E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice . Mol Cell Biol 1996 ; 16 : 5058 – 5068 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Cheon S , Poon R, Yu C et al. Prolonged beta-catenin stabilization and TCF-dependent transcriptional activation in hyperplastic cutaneous wounds . Lab Invest 2005 ; 85 : 416 – 425 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: S.A-N.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; D.G. and H.W.: collection and assembly of data, final approval of manuscript; C.B.: collection of data, final approval of manuscript; C.K.: provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript; B.A.A.: conception and design, provision of study material, assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. Disclosure of potential conflicts of interest is found at the end of this article. First published online in STEM CELLSEXPRESS July 7, 2011. Telephone: 416-813-7980; Fax: 416-813-6414 Copyright © 2011 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Pax7 Expressing Cells Contribute to Dermal Wound Repair, Regulating Scar Size through a β-Catenin Mediated Process
JF - Stem Cells
DO - 10.1002/stem.688
DA - 2011-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/pax7-expressing-cells-contribute-to-dermal-wound-repair-regulating-4gJBv49nkY
SP - 1371
EP - 1379
VL - 29
IS - 9
DP - DeepDyve
ER -