Fig 2: Papillary and reticular fibroblast lineages give rise to Ng2+ perivascular populations during wound healing. (A) Lrig1CreER; tdTomato mouse line with labeling and wounding strategy. (B) Representative immunofluorescence images of skin from Lrig1CreER; tdTomato mice treated with tamoxifen at E18.5 and isolated at 10 days post wounding labeled with Cd31 (green), Ng2 (gray) and DAPI nuclear counterstain (blue). The panel on the right-hand side is a higher magnification image of part of boxed region on the left-hand side. White arrows indicate Lrig1CreER tdTomato blood vessel resident perivascular cells expressing Ng2. (C) The percentage of Cd31 positive blood vessels within and outside the wound bed with Lrig1 tdTomato positive Ng2 expressing cells at 10 days post wounding. (D) Graph showing no significant difference in the percentage of Cd31 positive blood vessels with Lrig1CreER tdTomato and Ng2 expression in papillary and reticular layers of the regenerating wound bed dermis. (E) Dlk1CreER; tdTomato mouse line with labeling and wounding strategy. (F) Representative immunofluorescence images of Dlk1CreER; tdTomato lineage traced mouse back skin treated at E18.5 and isolated at 10 days post wounding labeled with Cd31 (green), Ng2 (gray) and DAPI nuclear counterstain (blue). The panel located on the right-hand side is a higher magnification image of part of the boxed region on the left-hand side. White arrows indicate Dlk1CreER tdTomato blood vessel resident cells expressing Ng2. (G) Graph depicting the percentage of blood vessels within and outside the wound bed with Dlk1 tdTomato Ng2 expressing cells. (H) Graph showing significantly higher percentage of Cd31 positive blood vessels with Dlk1 tdTomato and Ng2 expression in the reticular layer of the regenerating wound bed dermis when compared to the papillary layer. Statistical analysis was paired T-test. Error bars represent ±SD. N = 3 mice (3 sections per mouse) were quantified. Scale bars 100 or 50 µm (higher magnification views).

Fig 3: Schematic summary showing origins of Ng2+ skin pericytes in unwounded and wounded skin. In unwounded skin, Lrig1 (green), and Ng2 (gray) lineages, labeled at E18.5, give rise to Ng2+ blood vessel resident pericytes in the papillary layer of the dermis whilst Dlk1 (red) and Ng2 lineages give rise to Ng2+ blood vessel resident pericytes in the reticular layer of the dermis. These NG2+ pericytes can differ in their Pdgfr status. In unwounded skin Lrig1 and Dlk1 fibroblast lineages contribute to Ng2+ pericyte populations in a spatially restricted manner. However, in wounded skin, the Lrig1 and NG2 lineages can contribute to pericytes in both layers of the dermis whilst the contribution of the Dlk1 lineage remains spatially restricted to the reticular layer.

Fig 4: Deletion of Smoothened in fibroblasts affects EF formation.K14?Nß-cateninER mice were examined in telogen or following 4OHT treatment to induce ectopic hair follicles (EFs) in combination with heterozygous Smoothened deletion via Dermo1Cre (Smo?Dermo/+) or homozygous Smoothened deletion via PdgfraCreER (Smo?Pdgfra). (a–c) H&E (a) and immunofluorescent staining (b,c) of dorsal skin sections. In b,c, sections were labelled with antibodies detecting GFP and K14. 4,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei. (c) Higher magnification images of b. (d) Representative flow cytometry plots of Lrig1+ fibroblasts after gating on PDGFRaH2BeGFP+ cells. (e–g) Summary of flow cytometric analysis. Data represent mean±s.e.m. (n=3). *,#P=0.05; **P=0.005 by a one-way analysis of variance, compared with telogen or EF skin, respectively. (h) Immunofluorescent staining with antibodies detecting Caveolin and K14, counterstained with DAPI. (i) Herovici staining. Scale bars, 200 µm.

Fig 5: Epidermal ß-catenin activation induces Hh and TGF-ß signalling in distinct fibroblast lineages(a,b) Immunolabelling (a) and Herovici staining (b) of skin from K14?Nß-cateninER mice in telogen, following 4OHT treatment to induce ectopic hair follicles (EFs) or in combination with heterozygous Smoothened deletion via Blimp1Cre (Smo?Blimp1/+). Scale bars, 200 µm. (c) Representative flow cytometry plots of Sca1+ and Lrig1+ fibroblasts after gating on PDGFRaH2BeGFP+ cells. (d–f) Summary of flow cytometric analysis. Data represent mean±s.e.m. (n=3). *,#P=0.05; ***,###P=0.0005 by a one-way analysis of variance, compared with telogen or EF skin, respectively. (g–i) Labelling Lrig1+ cells. (g) Experimental strategy; (h,i) skin sections from adult mice treated with vehicle (h) or RepSox (i). Insets are higher magnification images. (j–n) Labelling Dlk1+ cells. (j) Experimental strategy; (k–n) skin sections from adult mice treated with vehicle (k,m) or RepSox (l,n). (m,n) Grey scale images with higher magnification of the red channel in e,f, respectively. AD, adipocyte layer; PC, panniculus carnosus. Dotted lines denote thickness of adipocyte layer. Scale bars, 200 µm. (o,p) Quantification of tdTomato+ cells within the interfollicular dermis (IFD) and AD of skin treated with RepSox or vehicle in Lrig1CreER (o) or Dlk1CreER (p) transgenic mice shown in g–n. Data represent mean±s.e.m. (n=3; 3 fields quantified per biological sample). *P=0.05, ***P=0.0005 by Student's t-test. (q–s) Relative expression of TGF-ß (q) and Hh target genes (s) and TGF-ß receptors (TGFBR; r) in Sca1- and Sca1+ (q) or Lrig1+ and Lrig1- (r,s) fibroblasts in adult dermis. Data represent mean±s.e.m. (n=3–4). Data were normalized to Sca1- or Lrig1+ cells in telogen. *P=0.05, **P=0.005 by Student's t-test. (t,u) Relative expression of Hh target genes (t) and TGF-ß and FGF target genes (u) in Sca1+ and Sca1- fibroblasts isolated from K14?Nß-cateninER/PDGFRaH2BeGFP double transgenic mice treated with 4OHT once or up to seven times. Data represent mean±s.e.m. (n=2; pooled from 3 to 5 mice).