As fluorescent labeling of the epithelial cell boundaries is key for quantitative analysis of these dynamic events, any fluorescently tagged membrane marker expressed in epidermal cells can be used in combination with the Et(Gal4-VP16) zc1044A Tg (UAS: nfsB-mCherry) zebrafish line required for inducing epithelial cell extrusion. The protocol below describes the specific steps for inducing extrusion and imaging 4 days post-fertilization (4dpf) Et(Gal4-VP16) zc1044A Tg (UAS: nfsB-mCherry) zebrafish larvae in combination with the Tg(UAS:Lifeact-GFP) utm1 transgenic line ( Eisenhoffer et al., 2017) to fluorescently label for F-actin in the surface epithelial cells. The damaged cells are then eliminated by extrusion ( Atieh et al., 2021), a process by which neighboring cells form a contractile actomyosin ring and eject the cell from the tissue while sealing the hole from its exit ( Rosenblatt et al., 2001). Upon exogenous addition of metronidazole (MTZ) to the plate or well containing the larvae, mCherry fluorescent epithelial cells expressing NTR will convert the MTZ to a cytotoxic byproduct that promotes DNA damage ( Curado et al., 2008). This is accomplished by expression of the genetically encoded enzyme nfsB (also referred to as nitroreductase, or NTR) fused to mCherry ( Davison et al., 2007) in the surface epithelial cells using the GAL4 enhancer trap line zc1044A ( Eisenhoffer et al., 2017). Here, we describe the procedure of inducing damage in a subset of cells within the outer epidermis of zebrafish larvae and methods for time-lapse imaging to track the dynamic remodeling and mechanical alterations within the tissue preceding epithelial cell extrusion.
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