Differential expression of villin and advillin by neuroendocrine and tuft cells in the murine lower airways

Previous studies identified a rare cell type in the mouse tracheal epithelium with immunoreactivity to the microvillus protein villin (Vil1), which persisted in mice lacking tuft cells due to deletion of the transcription factor Pou2f3. This study aimed to clarify the identity of this ill-defined cell type. Ultrastructurally, all cells with tightly packed microvilli observed in the tracheal epithelium of Pou2f3^-/--mice contained basally located dense core vesicles, a characteristic feature of neuroendocrine cells (NEC). Accordingly, immunofluorescence double-labeling utilizing NEC markers revealed villin-labeling in two thirds of NEC in the trachea, a reporter mouse strain showed Cre recombinase activity driven by the Vil1 promoter in a subpopulation of tracheal NEC, and analysis of single cell RNA sequencing data revealed Vil1-mRNA expression by tracheal NEC. Notably, only a minimal fraction (≈1%) of bronchopulmonary NEC (solitary and clustered in neuroepithelial bodies) displayed villin-immunoreactivity, despite nearly half of them having a history of Vil1 promoter activity. Microvilli of tuft cells differed ultrastructurally from those of NEC, and the majority of tuft cells were immunoreactive to advillin (Avil), showed Avil promoter activity as indicated by a reporter mouse strain, and expressed Avil-mRNA in the sequencing data set. This study uncovers villin-expressing cells in the lower airways as a cell population hidden among NEC. Advillin, not villin, is identified as a marker for airway tuft cells. This should be considered in interpreting findings based on the use of villin as a marker or Cre-driver when investigating rare cells in the murine airways.