Wound repair is controlled temporally and spatially to restore tissue homeostasis. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and wound healing compared to tail transection (LeBert et al., 2018). Here we characterize different injury models in larval zebrafish to dissect temporal and spatial dynamics of repair in complex damage. We found that each damage model triggers distinct inflammatory and tissue responses, with Stat3 and TGFβ playing key roles in the regulation of mesenchymal cells during simple repair. While thermal injury disrupts collagen fibers initially, healing is recovered as inflammation resolves, and mesenchymal cells and collagen fibers align. By contrast, infected wounds lead to persistent inflammation and loss of mesenchymal cells, resulting in minimal tissue repair. These wound models have broad physiological relevance, thereby providing a valuable advance in our toolkit to probe the dynamics of inflammation and wound repair in complex tissue damage.

Acute and chronic injuries are characterized by leukocyte infiltration into tissues. Although matrix metalloproteinase 9 (Mmp9) has been implicated in both conditions, its role in wound repair remains unclear. We previously reported a zebrafish chronic inflammation mutant caused by an insertion in the hepatocyte growth factor activator inhibitor gene 1 (hai1; also known as spint1) that is characterized by epithelial extrusions and neutrophil infiltration into the fin. Here, we performed a microarray analysis and found increased inflammatory gene expression in the mutant larvae, including a marked increase in mmp9 expression. Depletion of mmp9 partially rescued the chronic inflammation and epithelial phenotypes, in addition to restoring collagen fiber organization, as detected by second-harmonic generation imaging. Additionally, we found that acute wounding induces epithelial cell mmp9 expression and is associated with a thickening of collagen fibers. Interestingly, depletion of mmp9 impaired this collagen fiber reorganization. Moreover, mmp9 depletion impaired tissue regeneration after tail transection, implicating Mmp9 in acute wound repair. Thus, Mmp9 regulates both acute and chronic tissue damage and plays an essential role in collagen reorganization during wound repair.

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.

Tissue injury leads to early wound-associated reactive oxygen species (ROS) production that mediate tissue regeneration. To identify mechanisms that function downstream of redox signals that modulate regeneration, a vimentin reporter of mesenchymal cells was generated by driving GFP from the vimentin promoter in zebrafish. Early redox signaling mediated vimentin reporter activity at the wound margin. Moreover, both ROS and vimentin were necessary for collagen production and reorganization into projections at the leading edge of the wound. Second harmonic generation time-lapse imaging revealed that the collagen projections were associated with dynamic epithelial extensions at the wound edge during wound repair. Perturbing collagen organization by burn wound disrupted epithelial projections and subsequent wound healing. Taken together our findings suggest that ROS and vimentin integrate early wound signals to orchestrate the formation of collagen-based projections that guide regenerative growth during efficient wound repair.

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.