EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasionGraphical abstract Highlights d Inducible epithelial cell loss allows modeling of fungal infection in zebrafish larvae d Extrusion of numerous cells exposes laminin and increases fungal adhesion and invasion d Cell extrusion stimulates epigen, mmp13a, and il1b expression and neutrophil recruitment d rhEPGN suppresses extrusion to provide protective effects against fungal invasion

Facial development requires a complex and coordinated series of cellular events, that when perturbed, can lead to structural birth defects. A quantitative approach to quickly assess morphological changes could address how genetic or environmental inputs lead to differences in facial shape and promote malformations. Here we report on a method to rapidly analyze craniofacial development in zebrafish embryos using Facial Analytics based on a Coordinate Extrapolation system, termed zFACE. Confocal images capture facial structures and morphometric data is quantified based on anatomical landmarks present during development. The quantitative morphometric data can detect phenotypic variation and informs on changes in facial morphology. We applied this approach to show that loss of smarca4a in developing zebrafish leads to craniofacial anomalies, microcephaly and alterations in brain morphology. These changes are characteristic of Coffin-Siris syndrome (CSS), a rare human genetic disorder associated with mutations in SMARCA4. Multivariate analysis of zFACE data facilitated the classification of smarca4a mutants based on changes in specific phenotypic characteristics. Together, zFACE provides a way to rapidly and quantitatively assess the impact of genetic alterations on craniofacial development in zebrafish.

Facial development requires a complex and coordinated series of cellular events, that when perturbed, can lead to structural birth defects. A standardized quantitative approach to quickly assess morphological changes could address how genetic or environmental inputs lead to differences in facial development. Here we report on a method to rapidly analyze craniofacial development in zebrafish embryos that combines a simple staining and mounting paradigm with Facial Analytics based on a Coordinate Extrapolation system, termed zFACE. Confocal imaging of frontal/rostral mounted embryos generates high-resolution images to capture facial structures and morphometric data is quantified based on a coordinate system that assesses 26 anatomical landmarks present at defined times in development. The semi-automated analysis can be applied to embryos at different stages of development and quantitative morphometric data can detect subtle phenotypic variation. Shape analysis can also be performed with the coordinate data to inform on global changes in facial morphology. We applied this new approach to show that loss of smarca4a in developing zebrafish leads to craniofacial anomalies, microcephaly and alterations in brain morphology. These changes are characteristic of humans with Coffin-Siris syndrome (CSS), a rare genetic disorder associated with mutations in SMARCA4 that is defined by anomalies in head size, intellectual disabilities and craniofacial abnormalities. We observed that smarca4a is expressed in craniofacial tissues and our multivariate analysis facilitated the classification of smarca4a mutants based on changes in specific phenotypic characteristics. Together, our approach provides a way to rapidly and quantitatively assess the impact of genetic alterations on craniofacial development in zebrafish.

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BackgroundEfficient animal models are needed in order to investigate fungal pathogenicity and antifungal therapy in the context of epithelial injury, e.g., due to anticancer chemotherapy. Using a Gal4 enhancer trap (GET) zebrafish line facilitating metronidazole (MTZ)-inducible ablation of epithelial (periderm) cells, we aimed to establish a mucositis model predisposing larvae for fungal invasion.Methods4 days post-fertilization Et(Gal4-VP16)zc1044A;Tg(UAS-1b:nfsB-mCherry)c264 zebrafish larvae were exposed to 10 mM MTZ in E3 medium for 5 h. After washing, larvae were incubated in Candida albicans or Rhizopus arrhizus spore suspensions (5 × 105–5 × 107/mL) for 16 h. Thereafter, larvae were washed again and survival was monitored until 72 h post-infection. Fungal burden was assessed by 18S qPCR and histopathology. For drug protection studies, 5 µg/mL amphotericin B (AMB) or posaconazole (PCZ) was added to the medium of R. arrhizus-infected larvae at 16 h post-infection.ResultsIn MTZ-treated GET larvae, inoculum-dependent mortality was found for both R. arrhizus (panel A) and C. albicans (panel B). High inter-experiment reproducibility of survival rates was seen (CV < 0.3). Using a GFP-expressing R. arrhizus strain, fungal invasion of the larval tissue was verified by fluorescence microscopy (panel C). PCZ and AMB improved survival rates of R. arrhizus-infected (5 × 106/mL) larvae from 46% to 85% and 51% to 86%, respectively (P < 0.001). Similarly, significantly reduced fungal burden in AMB and PCZ-treated larvae was documented by qPCR (panel D) and histopathology. In additional validation experiments, the hypo-virulent phenotypes of a CotH-depleted R. arrhizus strain and filamentation-defective C. albicans mutants (Δefg1 and Δcph1) were recapitulated in zebrafish larvae with epithelial cell loss.ConclusionWe have established a robust and reliable model of invasive mycoses by controlled ablation of epithelial cells in zebrafish larvae, allowing for rapid immersion-based interrogation of different infection and treatment options. Our proof-of-concept experiments suggest that GET zebrafish larvae are positioned as an appealing high-throughput in vivo system for antifungal drug screening or comparative virulence studies. Disclosures All authors: No reported disclosures.