Distinct fibroblast lineages determine dermal architecture in skin development and repair

Abstract

Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibres of the extracellular matrix (ECM). Even within a single tissue, fibroblasts exhibit considerable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing in mice, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle, which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesize the bulk of the fibrillar ECM, and the preadipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialization. Epidermal β-catenin activation stimulates the expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

Figure 1. Morphological and molecular markers of embryonic and postnatal fibroblasts

(a-c) Sections of mouse back skin. (a) H&E staining. P: papillary dermis; R: reticular dermis; H: hypodermis; P: panniculus carnosus; DP: dermal papilla. (b) 10μm sections immunostained with PDGFRa antibodies and secondary antibody control with DAPI nuclear counterstain. Red squares show areas digitally sampled for the tissue screen. (c) Tissue screen of upper and lower dermis. 3 biological replicates for each stain were performed. Images are representative samples of sections shown Fig. E1, E2. Scale bars: 50 μm.

Figure 2. Skin reconstitution assays

(a) Experimental set up for (b-f). (b-c) Grafts immunostained with DAPI counterstain (blue). SG: sebaceous gland. Arrows: APM. (d-f) Contribution of PDGFRaH2BeGFP cells to dermal compartments. (g) Experimental set up for (h-k). (h) Macroscopic views of grafts. (i) Contribution of GFP+ cells to grafts. Arrows: GFP+ DP. (j) Hair follicles per graft. (k) GFP+ DP per 120 microns graft. N=3 biological replicates per experiment. *P <0.05; **P <0.005. Horizontal whole mounts shown. Scale bars: (b-c) 40 μm (e) 30 μm (h) 2.5mm (i) 50 μm.

Figure 3. Fibroblast lineage commitment during skin development

(a, e, i, p, q) Labelling strategies. (b, c, f, g, j, l-o, r-t) Contribution of labelled cells to different dermal compartments. AP: arrector pili muscle; DP: dermal papilla; Adipo: adipocyte. (d, h, k, u) Average number labeled cells per unit area of dermis (defined by hair follicle spacing). (v, w) Schematic summary of lineage restriction. 3 biological replicates analyzed per experiment. Sections counterstained with DAPI. NS – Not statistically significant different; **P <0.005, ***P <0.0005. Scale bars: 50 μm.

Figure 4. Contribution of fibroblast lineages in adult skin

(a, d, g, l) Labelling strategies. (b, c, e, f, h, m, n, q) Histological analysis of horizontal whole mounts. (i-k) Flow cytometric quantification of fibroblast markers. (o, p) Quantitation of ectopic hair follicles in (o) 17 day wound bed whole mount sections and (p) entire 24 day wound bed. (i-k) n = 3, (o, p) n = 6 biological replicates. *P <0.05, **P <0.005, ***P <0.0005. Scale bars: 200μm (b, c, e, f), 100μm (m, n, q) or 50μm (higher magnification views).