Reprogramming fibroblasts embryonic origins to overcome skin fibrosis and scarring. - 7. Project Summary/Abstract Adult human skin heals by developing fibrotic scar tissue, which can result in devastating disfigurement, growth restriction, and permanent functional loss. Despite a plethora of clinical options, no current treatment strategies successfully prevent or reverse this fibrotic process, and over $20 billion is spent each year in the United States for the treatment of scars and their sequelae. Fibroblasts are recognized as the primary cell responsible for depositing extracellular matrix and causing skin fibrosis. However, progress towards the development of treatments aimed at reducing scars is impeded by a limited understanding of specific fibroblast subpopulations responsible for regenerative healing. We have observed that neural crest-derived facial skin wounds heal with less fibrosis than mesoderm-derived scalp wounds, somite-derived dorsal wounds, and lateral plate mesoderm- derived ventral wounds. Furthermore, through single cell RNA sequencing, we have identified that neural crest- derived facial fibroblasts promote regeneration following skin injury through a Robo2-EID1-EP300 axis, and bromodomain inhibition of EP300 guides fibroblasts to heal with reduced skin scarring. In this proposal, we examine the potential of the Robo2-Eid1-EP300 axis and EP300 bromodomain inhibition to guide dorsal scarring fibroblasts to heal with reduced fibrosis. We will employ both cell transplantation and CRISPR-Cas9 approaches, using histology, immunohistochemistry, transcriptional analysis, and flow cytometry to evaluate the regenerative capacity of Robo2+ fibroblasts within wounds. We will then determine the role of EP300 interacting inhibitor of differentiation 1 (EID1) in regulating Robo2 fibroblast activity in skin wounds. We will employ cell transplantation and CRISPR-Cas9 approaches to robustly determine whether activation of EID1 promotes regeneration of dorsal wounds to heal like facial wounds. Finally, having established that the Robo2-EID1-EP300 axis is responsible for regenerative healing, we will inhibit EP300 signaling using both small molecule and transgenic approaches to guide facial-like regenerative healing in dorsal wounds. Our ultimate translational goal is to develop therapeutics that target fibrogenic wound cell dynamics to promote regenerative healing. Collectively, the proposed work will significantly enhance our understanding of the key molecular and cellular determinants of cutaneous scarring, shedding light on the contributions of Robo2-EID1-EP300 activity in wound repair and scarring, and inform the development of novel anti-scarring therapeutics.
