Skin wounds generally heal by scarring, a fibrotic process mediated by the Engrailed-1 (En1) fibroblast lineage. Scars differ from normal unwounded skin in three ways: (i) They lack hair follicles, sebaceous glands, and other dermal appendages; (ii) they contain dense, parallel extracellular matrix fibers rather than the “basket-weave” pattern of uninjured skin; and (iii) as a result of this altered matrix structure, they lack skin’s normal flexibility and strength. A successful scar therapy would address these three differences by promoting regrowth of dermal appendages, reestablishment of normal matrix ultrastructure, and restoration of mechanical robustness. However, little is known about the cellular and molecular mechanisms blocking a regenerative healing response in postnatal skin, or whether these mechanisms can be bypassed by modulating specific fibroblast lineages.

We asked whether scarring fibroblasts are derived purely from expansion of existing En1 lineage–positive fibroblasts present in unwounded skin, or whether En1 scar fibroblasts could arise de novo by activation of En1 expression in postnatal, En1 lineage–negative fibroblasts within the wound niche. We used fibroblast transplantation as well as transgenic mouse models to trace En1 expression in a spatiotemporally defined fashion. Next, we studied fibroblast responses to mechanical forces in vitro and in vivo to establish a mechanotransduction mechanism linking skin tension to postnatal En1 expression. Finally, we used chemical (verteporfin) and transgenic inhibition of mechanotransduction signaling [diphtheria toxin ablation of En1-expressing fibroblasts, floxed Yes-associated protein (YAP) knockout] to modulate En1 expression during wound healing. Experimental wounds were compared to unwounded skin and scars (phosphate-buffered saline control) by RNA sequencing, quantitative histopathological comparison (using a custom image-processing algorithm), and mechanical strength testing.

Fibroblast transplantation and lineage-tracing studies reveal that En1 lineage–negative fibroblasts (ENFs) of the reticular (deep) dermis activate En1 in the wound environment, generating ~40 to 50% of scar fibroblasts. This phenomenon depends on mechanical cues: ENFs cultured on soft substrates or treated with chemical inhibitors of mechanical signaling proteins (e.g., YAP) do not activate En1. Comparison of ENFs with En1-expressing and En1 knockdown (short hairpin RNA) fibroblasts by RNA sequencing suggests that En1 regulates a wide array of genes related to skin fibrosis. In healing wounds, YAP inhibition by verteporfin blocks En1 activation and promotes ENF-mediated repair, yielding skin regeneration in 30 days with recovery of functional hair follicles and sebaceous glands. Quantitative comparison of scars and regenerated skin shows that YAP inhibition induces recovery of normal dermal ultrastructure, which in turn confers restoration of normal mechanical breaking strength. Diphtheria toxin–mediated ablation of postnatal En1-expressing fibroblasts and fibroblast-targeted transgenic YAP knockout similarly promoted recovery of normal skin structures, which suggests that modulation of En1 activation, whether direct or indirect, can yield wound regeneration.

By delineating how physical stimuli provoke ENFs to contribute to fibrosis, we identify YAP and En1 as possible molecular targets to prevent scarring. Furthermore, we have shown that inhibition of YAP signaling prevents En1 activation during wound healing, thus encouraging ENF-mediated wound repair without fibrosis and with regeneration of secondary skin …