Skin fibroblast lineage tracing reveals layer-specific cooperation in wound repair

Skin fibroblasts exhibit remarkable heterogeneity across their multilayered cutaneous architecture, yet their roles during wound repair remain elusive, mainly due to the lack of layer-specific genetic tools. Here, we employed a dual-recombinase strategy to develop genetic lineage-tracing tools for all five layers of skin fibroblasts: papillary, reticular, hypodermal, panniculus carnosus (PC), and fascia fibroblasts. Our study revealed an intricate spatiotemporal pattern of fibroblast contributions across different wound severities. Temporally, deep wounds elicited a sequential contribution of these fibroblasts in three stages, while superficial wounds involved two. The early-contributing fibroblasts predominantly occupied the wound bed during the closure stage, while subsequently recruited populations mainly entered during tissue remodeling and scar formation. Spatially, as remodeling progressed, fibroblasts reorganized into a hierarchical arrangement closely mirroring their pre-injury spatial distribution. Our study elucidates the coordinated spatiotemporal interplay of fibroblasts during wound repair and may spur novel therapeutic modalities and interventions for cutaneous healing.