Biology of Submucosal Gland Stem Cells in the Airway

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Project Summary Airway submucosal glands (SMGs) are known to contain reserve stem cells for the surface airway epithelium (SAE). In mice, this niche serves only the trachea; however, in larger mammals such as humans, pigs and ferrets, SMGs are present throughout the intralobar cartilaginous airways and may serve the broader function of maintaining the proximal conducting airway epithelium in the setting of disease. During the previous six funding cycles, this grant has used mouse and ferret genetic models to address multiple aspects of airway SMG biology, SMG stem/progenitor cell biology, and cystic fibrosis (CF) lung pathogenesis. This proposal aims to identify the subpopulations of glandular myoepithelial cells (GMECs) that participate in airway repair, as well as the Wnt- regulated mechanisms that control their behavior following injury. Based on our preliminary data, we hypothesize that Lef-1 and Sox9 transcription factors differentially control Wnt-responsive GMEC states that orchestrate the commitment, renewal, migration, and proliferative expansion of GMECs on the airway surface. Aim 1 will define the biology of tracheal GMECs in mice and utilize an array of transgenic lines (intersectional lineage tracing, conditional knockout, Wnt-reporters, Dox-inducible H2B-GFP) to study the involvement of Lef-1 and Sox9 in regulating processes that control GMEC commitment, renewal, migration, and proliferation. Aim 2 will use intersectional lineage tracing in ferrets to define the participation of GMEC subtypes and gland ductal cells in maintaining the extralobar and intralobar SAE at homeostasis and following injury. We hypothesize that gland ducts are a site for GMEC maturation to a pre-basal cell state, and that this underlying hierarchical relationship is disturbed in chronic airway diseases such as CF. Aim 3 will test this hypothesis by identifying disturbances in GMEC and gland duct niches in the setting of mild and severe CF lung disease, using VX-770-responsive CFTRG551D ferrets. Novel aspects of these studies include the first non-rodent fate mapping in a species (ferret) that closely models CF lung disease and SMG biology of humans, and supporting fate mapping data demonstrating that ferret GMECs (ACTA2CreER) and gland ductal cells (KRT7CreER) participate in SAE repair. This research will also shed light on differences in the behavior of SMG stem cell compartments in the extralobar and intralobar cartilaginous airways, which cannot be addressed in mice because they lack SMGs in the intralobar airways. This project is designed to enhance our understanding of stem cell phenotypes in airway SMGs and the mechanisms that regulate their participation in SMG and surface airway repair. Given that GMECs can regenerate both glandular and surface airway cell types, they are an attractive target for gene editing in CF and such efforts will be enhanced by knowledge gained from this proposal. Furthermore, this work will delineate disease-associated changes to SMG stem cell niches that may be important for the pathogenesis of CF airway disease and other hypersecretory diseases that affect SMGs, such as asthma and chronic bronchitis.