A protocol to evaluate epithelial regeneration after inducing cell loss in zebrafish larvae.

Epithelial tissues sustain barrier function by removing and replacing aberrant or unfit cells. Here, we describe approaches to evaluate epithelial restorative capacity after inducing cell loss in zebrafish larvae. We provide details to quantify morphological changes to the tail fin epithelium after cell loss, and instructions to interrogate changes in gene expression and proliferation associated with replacement of the lost cells. Together, this approach establishes an in vivo vertebrate model for the rapid assessment of molecular pathways controlling epithelial regeneration. For complete details on the use and execution of this profile, please refer to Wurster et al. (2021).

EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion.

Severe and often fatal opportunistic fungal infections arise frequently following mucosal damage caused by trauma or cytotoxic chemotherapy. Interaction of fungal pathogens with epithelial cells that comprise mucosae is a key early event associated with invasion, and, therefore, enhancing epithelial defense mechanisms may mitigate infection. Here, we establish a model of mold and yeast infection mediated by inducible epithelial cell loss in larval zebrafish. Epithelial cell loss by extrusion promotes exposure of laminin associated with increased fungal attachment, invasion, and larval lethality, whereas fungi defective in adherence or filamentation have reduced virulence. Transcriptional profiling identifies significant upregulation of the epidermal growth factor receptor ligand epigen (EPGN) upon mucosal damage. Treatment with recombinant human EPGN suppresses epithelial cell extrusion, leading to reduced fungal invasion and significantly enhanced survival. These data support the concept of augmenting epithelial restorative capacity to attenuate pathogenic invasion of fungi associated with human disease.

Stem cell proliferation is induced by apoptotic bodies from dying cells during epithelial tissue maintenance.

Epithelial tissues require the removal and replacement of damaged cells to sustain a functional barrier. Dying cells provide instructive cues that can influence surrounding cells to proliferate, but how these signals are transmitted to their healthy neighbors to control cellular behaviors during tissue homeostasis remains poorly understood. Here we show that dying stem cells facilitate communication with adjacent stem cells by caspase-dependent production of Wnt8a-containing apoptotic bodies to drive cellular turnover in living epithelia. Basal stem cells engulf apoptotic bodies, activate Wnt signaling, and are stimulated to divide to maintain tissue-wide cell numbers. Inhibition of either cell death or Wnt signaling eliminated the apoptosis-induced cell division, while overexpression of Wnt8a signaling combined with induced cell death led to an expansion of the stem cell population. We conclude that ingestion of apoptotic bodies represents a regulatory mechanism linking death and division to maintain overall stem cell numbers and epithelial tissue homeostasis.