["The Journal of Physiology, Volume 604, Issue 7, Page 2642-2658, 1 April 2026. ", "\nAbstract figure legend A genetic approach to study ionocyte function. An animal expressing tamoxifen‐inducible Cre recombinase driven by the achaete scute‐like 3 (Ascl3) gene locus was produced (Ascl3P2‐GCE). A, this animal was crossed with a floxed Tomato fluorescent protein reporter strain (R26TdT) animal to facilitate identification of Ascl3+ ionocytes and to study the complement of proteins expressed in these cells. B, the function of Ascl3+ ionocytes in salivary glands was studied by crossing Ascl3P2‐GCE animals with floxed diphtheria toxin fragment A animals (R26DTA) to ablate Ascl3+ ionocytes. C, animals that expressed a genetically encoded Ca2+ indicator in Ascl3+ ionocytes were produced by crossing Ascl3P2‐GCE animals with floxed R26GCaMP6f animals to enable the study of ionocyte intracellular Ca2+ dynamics in vivo using multiphoton microscopy. D, in vivo Ca2+ dynamics were compared in ionocytes and other salivary gland cells by producing an animal which expresses genetically encoded Ca2+ indicator in essentially all salivary gland resident cells.\n\n\n\n\n\n\n\n\n\nAbstract\nIonocytes are distinct epithelial sensory cells scattered throughout the ductal system of the salivary glands. These cells are distinguished by their unusual morphology, as well as by a specific transcriptomic signature that includes the expression of the forkhead boxI1 (Foxi1) and achaete scute‐like 3 (Ascl3) transcription factors. Currently little is known about the biology or function of ionocytes in the salivary glands. To facilitate the characterization of these cells, we produced an inducible Cre mouse allele driven by the Ascl3 promoter. This strain was crossed with a reporter to fluorescently label Ascl3+ ionocytes, highlighting that they are the site of enriched cystic fibrosis transmembrane conductance regulator (Cftr) expression in the salivary glands and demonstrating the proximity of these cells to neurons. Conditional Cre‐mediated cell ablation, using diphtheria toxin (DTA), removed Ascl3+ ionocytes from the salivary glands and resulted in an altered pH of total saliva, supporting a function for ionocytes in transepithelial ion flow. Finally Cre‐mediated expression of the calcium indicator GCaMP6f revealed thatAscl3+ ionocytes exhibit unique properties not observed in the acinar or surrounding duct cells, including elevated basal [Ca2+]i, spontaneous blinking in the absence of stimulation and a rapid loss of [Ca2+]i after nerve stimulation. These unique properties distinguish ionocytes as a specialized subset of salivary gland duct cells.\n\n\n\n\n\n\n\n\n\nKey points\n\nIonocytes are epithelial cells originally identified in marine teleosts and subsequently reported to be present in pulmonary epithelia, olfactory epithelium and salivary and lachrimal glands.\nWe produced an inducible Cre mouse allele driven by the Ascl3 promoter to study the distribution and function of Ascl3+ ionocytes in salivary glands.\nBy crossing this strain with a fluorescent reporter, we demonstrated the prominent localization of Ascl3+ ionocytes in salivary gland ducts.\nCre‐mediated expression of the genetically encoded Ca2+ indicator GCamp6f in Ascl3+ ionocytes revealed unique Ca2+ signalling properties of this cell type distinct from acinar cells and other ductal cell types.\nCre‐mediated ablation of Ascl3+ ionocytes altered the pH of the final saliva.\nWe conclude that ionocytes resident in salivary gland ducts function to modify the primary saliva contributing to the composition of the final saliva entering the oral cavity.\n\n\n"]