Exploring mechanisms of scar-free skin wound healing in adult zebrafish in comparison to mouse

Adult zebrafish have the ability to perfectly regenerate their skin after injury without leaving a scar behind. Yet, they intermediately form a collagen-rich granulation tissue that later fully regresses. In contrast, adult mammals lose this ability, resulting in persistent tissue fibrosis and scarring. We performed single-cell RNA sequencing and first HCR-based spatial transcriptomics to characterize the dynamics and heterogeneity of involved cell types during different stages of zebrafish cutaneous wound healing, focusing on macrophages and fibroblasts. Macrophage subclusters display pro-inflammatory and/or anti-inflammatory/pro-repair characteristics, and fibroblast subclusters characteristics of extracellular matrix formation and degradation, which largely co-exist during all stages of wound healing. Some wound-specific cells have a signature similar to that of myofibroblasts implicated in fibrotic healing in mammals. However, in contrast to mammalian myofibroblasts, they lack collagen expression, suggesting that they might only share the beneficial, but not the detrimental roles of their mammalian counterparts. Strikingly, zebrafish fibroblasts, in addition to expressing anti-fibrotic genes, express multiple genes with described pro-fibrotic effects in mammalian models. One of them is plod2, which encodes lysylhydroxylase 2. In cutaneous mouse wounds, Plod2 is induced in fibroblasts by the macrophage-released Resistin-like molecule RELMα encoded by the Retlna gene, promoting the formation of DHLNL collagen crosslinks and thereby less resolvable fibrotic tissue. retln genes are absent from the zebrafish genome; nevertheless, plod2 expression is initiated in zebrafish dermal fibroblasts upon wounding, in this case via TGFβ signaling, accompanied by increased collagen DHLNL crosslinking. Yet, both transgenic overexpression and genetic knock-out of plod2 do not interfere with granulation tissue formation and regression, pointing to additional pathways assuring the resolution of temporary fibrosis in zebrafish skin wounds even in the presence of strong collagen crosslinking.