Developmental origins of fibroblasts determine their scarring potential in wound healing

Summary: Abstract Body: Skin wounds at disparate body sites heal with varying degrees of scarring. The location-dependent fibroblast heterogeneity acquired during development points to cell-intrinsic properties determining fibroblasts’ scarring potential. To identify fibroblast signaling pathways governing anatomically variant fibrosis, we used a mouse injury model with excisional wounds of facial, scalp, ventral, and dorsal skin. Histological assessment at post-operative day (POD) 14 showed that facial and dorsal skin healed with the least and the most scarring, respectively. Single-cell RNA-seq of POD-14 fibroblasts from these sites revealed that facial fibroblasts highly expressed the neural crest development gene Robo2 and the histone acetyltransferase EP300 inhibitor gene Eid1. Dorsal wound transplantation of fibroblasts with CRISPR-mediated Robo2 or Eid1 overexpression led to reduced scar width, lower collagen deposition, less fibrotic extracellular matrix (ECM) ultrastructure, and increased neural crest marker levels compared to control dorsal wounds. Ep300 knockout in fibroblasts or small-molecule EP300 inhibition phenocopied the reduced scarring features of the transplantation experiments in dorsal wounds, and led to lower fibroblast H3K27 acetylation around ECM genes (e.g., Col1a1, Col3a1, Fbln). Thus, ROBO2-EID1-EP300 signaling underlies the reduced fibrotic potential of facial fibroblasts, and its activation in scar-prone fibroblasts suppresses their fibrotic activity by repressing ECM gene transcription. These data provide a mechanism for anatomically variant scarring, and highlight a novel strategy that manipulates the fibroblast-intrinsic fibrogenic potential for anti-scarring therapies. Dayan Li¹, Michelle Griffin¹, Kellen Chen², Jennifer Parker¹, Jason Guo¹, Michael Januszyk¹, Seungsoo Kim¹, Katerina Kraft¹, Mauricio Downer¹, Annah Morgan¹, Maxwell Kuhnert¹, Hanqi Yao¹, Serena Jing¹, Caleb Valencia¹, Asha Cotterell¹, Geoffrey Gurtner², Howard Chang¹, Joanna Wysocka¹, Derrick Wan¹, Michael Longaker¹ 1. Stanford Medicine, Stanford, CA, United States. 2. University of Arizona Medical Center, Tucson, AZ, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing