(658) Characterization of CFTR-expressing Epithelial Cell Lineages in Mouse Airways by Fate Mapping

Background: CFTR is heterogeneously expressed in distinct airway epithelial cell types such as secretory cells, ciliated cells, intermediate cells, and pulmonary ionocytes. However, it remains unclear how the diverse cellular patterns of CFTR expression coordinate physiologic homeostasis of airway epithelial function. Previous studies show pulmonary ionocytes express high levels of CFTR in both mouse and human airway epithelia. However, the specification and function of pulmonary ionocytes in airway epithelia remains unclear. To better define mechanisms of basal cell specification to CFTR expression cell type in the proximal airway, we generated a mouse model capable of fate mapping CFTR-expressing cell types.

Methods: We generated a Rosa26nTnG/Cftr-ires-CreERT2 (Rosa/CftrCre) reporter transgenic mouse line in which CreERT2 expression is dictated by the CFTR gene. CFTR-expressing cells in Rosa/CftrCre mice were genetically labeled by tamoxifen induction at different ages. The CFTR-expressing cell lineages were identified by immunolocalization of EGFP and airway epithelial cell markers in native mouse airway and using an air-liquid interface (ALI) cultures generated from primary surface tracheal basal cells and submucosal gland (SMG) cells of Rosa/CftrCre mouse airways.

Results: The transgenic Rosa/CftrCre mouse is a Rosa26nTnG Cre/LoxP reporter line containing the Cftr gene-derived ires-CreERT2 driver. CFTR-expressing cells were identified by the conversion of nuclear tdTomato (nT) to nuclear GFP (nG) following a tamoxifen induction. In the trachea of transgenic mouse, rare GFP-labeled cells were mainly confined to the proximal tracheal epithelia and SMGs. Labeled secretory and intermediate basal cells were absent in the trachea and only rare club labeled cells were present in the intralobar airways. Notably, high frequency of GFP+ Type II alveolar cells were observed lung parynchyma. In addition, pronounced GFP-labeled cells were present in the intestinal crypts of transgenic mice. A small subset of GFP-labeled cells were also observed in other tissues including stomach, testis in males, kidneys, and pancreas. Immunolocalization analysis demonstrated that majority of GFP-labeled cells also express ionocyte marker Bsnd and FoxI1 in the trachea, and surfactant protein-C in alveoli. The observation in native transgenic mouse trachea was further corroborated in ALI cultures generated from cells of surface airway epithelium (SAE) and SMGs. Fate mapping of CFTR expressing during airway epithelial cell differentiation in the presence of hydroxytamoxifen demonstrated that >80% of GFP-labeled lineages expressed ionocyte markers Bsnd and FoxI1, but not major epithelial cell types such as ciliated, secretory and basal cells. Notably, SMG-derived progenitors generated significantly more lineage-labeled cells expressing than SAE progenitors, the majority of which also expressed Bsnd and FoxI1, suggesting that SMGs may be enriched in progenitors of pulmonary ionocytes.

Conclusions: The transgenic Rosa/CftrCre mouse line is a useful tool to define the phenotypes and function of CFTR-expressing cells in airway epithelium. Further studies of the GFP-labeled cells at single cell resolution may help to better define pulmonary ionocyte progenitors in the SAE and SMGs.

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