Distinct fibroblast lineages determine dermal architecture in skin development and repair.

Methods

Transgenic mice
Mice were maintained on a C57/Blk6 and CBA F1 background. PDGFRaCreER28 or Dlk1CreERt (ICS) mice were bred with CAGCATeGFP29 or ROSA26tdTomato (Jax Labs - 007905). PDGFRaeH2BeGFP mice30 were obtained from Jackson Laboratories. Blimp1GFP14 and Blimp1Cre (Jax Labs - 008827) 13 mice have been described previously. K14ΔNβ-cateninER mice have previously been described. Tamoxifen (Sigma Aldrich) was injected intraperitoneally into adult mice. 2mg of Tamoxifen in 100ul of acetone was administered topically to the back skin of pups. Bone marrow reconstitution experiments and wounding (8 mm diameter full thickness wounds) were performed as described previously26. All experiments were performed according to the guidelines of the UK Government Animals (Scientific Procedures) Act 1986 and underwent ethical review by Cambridge University and King’s College London. Experiments were performed on female mice; however, the markers evaluated showed the same distribution in males. All experiments were performed on 3 or more female mice.

Flow cytometry
P2 disaggregated dermal preparations were prepared as previously described 11 and labelled with the antibodies listed below.

RT-PCR
This was performed as previously described27. Analysis was performed by the delta-Ct method. Primer probe sets were purchased from Applied Biosystems and used according to manufacturer’s recommendations: Ly6a/Sca1 - Mm00726565_s1; Dlk1/Pref1 - Mm00438422_m1; CD26/DPP4 - Mm00494538_m1; pparg -Mm01184322_m1; Zfp423 - Mm00473699_m1; Acta2 - Mm01546133_m1 Axin 2 – Mm01266783_m1; Dkk1 – Mm00438422_m1; Lef1 – MM01310389_m1; Tcf3 – Mm01188714_m1; Tcf7l2 – Mm00501505_m1; Wnt5a – Mm00437347_m1; Blimp1 – Mm01187285_m1.

Chamber grafting assay
This procedure was performed as previously described9. 8 × 106 wild type adult dorsal epidermal cells were combined with 5 × 106 wild type P2 dermal cells and 105 flow sorted PDGFRaH2BeGFP P2 dermal cells.

Histology and microscopy
5 μm frozen sections and horizontal whole mounts were stained with the antibodies listed. The tissue array (Fig. 1c) was constructed from digitally excised images of areas of upper and lower dermis (Fig. 1b). All microscopy was performed on a Leica SP5 or Nikon A1 confocal microscope and images were analyzed in Image J and Adobe Photoshop CS6.
Horizontal whole mounts were prepared as previously described11. Skin was collected and fixed in 4%PFA for 15-30 minutes then embedded in cryomolds. Sections were cut on a cryostat at a thickness of between 50-150 μm. Instead of attaching sections with OCT onto slides, forceps were used to place sections into 1xPBS at room temperature. The 1XPBS dissolved away the OCT and tissue was then ready for staining in 300-500μl of PB buffer (1XPBS containing 0.5% skim milk, 0.25% Cold Water Fish Skin Gelatin, 0.5%Triton X100) in Eppendorf tubes. Sections could be stored for 3-6 months in 1xPBS at 4°C. Staining is subsequently performed by incubation with primary antibody overnight at 4°C on a rocker. Secondary antibodies were incubated for at least 2 hours at room temperature after 1 wash in 1xPBS for 1 hour. The tissue was then mounted on coverslips with a small volume of 100% glycerol and analysed by confocal microscopy.

Antibodies
The following antibodies were used: Itga8 (R&D Systems AF4076), Dlk1 (R&D Systems AF1144), CD26 (R&D Systems AF954), Sca1(R&D Systems AF1226), PDGFRa (R&D Systems AF1062), Fabp4 (R&D Systems AF1443), AdipoQ (R&D Systems AF1119), Ephb3 (R&D Systems AF432), Lrig1 (R&D Systems AF3688), Podplanin (R&D Systems AF3244), Transglutaminase2 (R&D Systems AF4376), Blimp1 (eBioscience 14-5963), CD26 (eBioscience 45-0261), Sca1 (eBioscience 56-5981-82), PDGFRa (eBioscience 14-140182), eCadherin (eBioscience 17-1449), CD34 (eBioscience 17-0349-42), CD44 (eBioscience 130441), CD45 (eBioscience 12-0451), CD31 (eBioscience 25-0311-82), GFP (Invitrogen A11122), Lipidtox (Rockland H34476), tdTomato (MBL 600-401-379), Dlk1 (Millipore D187-5), ColIV (Cell Signaling AB8201), Lef1 (Cell Signaling 2330S), Vimentin (Cell Signaling 5741S). The alpha-smooth muscle actin antibody was a gift from Frank Nestle, King’s College London.

Clonal growth of fibroblast subpopulations in hydrogels
Flow sorted PDGFRa populations were seeded into Extracel hydrogels (Glycosan Biosystems, Salt Lake City, UT), which contain cross-linked gelatin and hyaluronic acid14, in individual wells of 24 well plates at a density of 5 × 104 cells per well. Cells were grown in Adipogenic Medium (R&D Systems) or standard Growth Medium (DMEM supplemented with 10% bovine serum) for 1 week. Cultures were fixed with 4% PFA, washed in PBS and stained with Lipidtox (Invitrogen 1:500) and DAPI. Spheres were scored positive for adipogenesis if they contained at least one lipid positive cell.

Graphing and Statistical Analysis
All graphs were generated using Excel, GraphPad Prism 6 and Adobe Illustrator CS4 software. Data are means ± standard error of the mean (SEM). A One way ANOVA parametric test was performed for experiments with P < 0.05 considered significant.
Sample size for animal experiments was determined on the basis of pilot experiments. In the case of the skin reconstitution assays, animals were excluded from analysis only if grafts were unsuccessful (i.e. chamber falling out 1-2 days after grafting).

Supplementary Material
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