Development of an original in vivo model to investigate real-time dynamics of skin repair

Summary: Abstract Body: Wound healing is a highly regulated process starting with re-epithelialization and concomitant immune cells recruitment, followed by tissue remodeling. Among animal models that should shed light this process, zebrafish is suitable thanks to its thin epidermis and dermis structures combined with its transparency, enabling live imaging. This study aimed to investigate the involvement and dynamics of the extracellular matrix in the various steps of skin repair using 4 days-post-fertilization zebrafish larvae. Transgenic fluorescent zebrafish lines were used. The larvae allowing the visualization of the basal keratinocytes’ plasma membrane and/or their cytoplasm, were crossed with lines in which neutrophils and macrophages are highlighted. Then, a laser-ablation technique was implemented to induce controlled wounds. Regardless of the lesion type, the re-epithelialization process exhibits 5 distinct phases. Real-time observations reveal actin-based cell movement of leader cells, cryptic lamellipodia formation in following cells, and a noticeable shift in cell-cell and cell-matrix contacts over distance. This is followed by the recruitment of neutrophils and macrophages at the injury site. Results reveal that Collagen XIV, a basement membrane-associated component, is also expressed by basal keratinocytes. Interestingly, lack of its expression in mutant fish impairs neutrophils recruitment at the injury site while increasing wound closure, revealing that extracellular matrix actively influences the immune response. Finally, immunostaining show that the dermis at the injury site contains disorganized collagen I fibrils and tenascin C extra-deposits, reminiscent to the granulation tissue formation. Hence, this original study highlights that zebrafish larvae, in conjunction with laser ablation and live imaging, provides a reliable model for investigating skin repair dynamics and drug testing while operating outside the scope of animal testing legislation. Hisoilat Bacar¹, Josselin Breugnot², Laure Delhon¹, Nicolas Goudemand¹, Elodie Aymard², Brigitte Closs², Maryline Malbouyres¹, Florence Ruggiero¹ 1. IGFL, UMR5242,ENS Lyon, Université Lyon 1, CNRS, Institut de Genomique Fonctionnelle de Lyon, Lyon, France. 2. SILAB, Brive, France. Stem Cell Biology, Tissue Regeneration and Wound Healing