Defining the lineage, mechanisms of maintenance, and function of a new injury-resistant airway epithelial structure: the hillock

We recently identified a novel airway epithelial structure, the hillock, in the murine trachea and we present evidence that similar structures occur in the human airway epithelium. Airway epithelial hillocks are stratified epithelial structures with multiple layers of cells that are distinct from the classic pseudostratified epithelium that lines most of the airway lumen. They possess novel polyploid luminal epithelial cells, but completely lack ciliated cells. We now show that these stratified hillock epithelial structures resist multiple forms of airway injury. We hypothesize that the stratified structure of hillock cells results in a protective barrier, in analogy to the protective function of the stratified epidermal cells that lie atop epidermal basal cells. Furthermore, we show that hillocks contain a unique population of basal stem cells marked by Cytokeratin 13 (KRT13). These basal cells are characterized by a very high baseline rate of turnover and are capable of rapidly migrating over denuded airway epithelium. We hypothesize that the diffuse distribution of injury-resistant airway hillocks facilitates disseminated migration of stem cells that have resisted severe injury. We show preliminary evidence that hillock basal stem cells, which are the first highly defined subset of airway basal cells in the murine trachea, are plastic and can produce normal pseudostratified epithelium after damage to recreate a normal epithelium. Finally, we show that murine hillocks increase in size and number with age. Interestingly, both smoking-induced squamous metaplasia and vitamin A-deficiency induced squamous metaplasia have both been associated with stratified KRT13+ epithelium suggesting they may originate from hillock-like cells. In this grant application, we first propose to define the lineage and plasticity of hillocks during homeostasis and after injury using 2 distinct genetic driver lines that are designed to specifically label hillocks and even more specifically hillock basal cells. We will also determine the functional significance of hillocks during epithelial homeostasis and after injury repair using diphtheria toxin- mediated ablation in combination with models of airway injury, with special attention focused on assessing the role of hillock polyploid cells. Using a combination of an explant model of murine tracheal regeneration, 2-photon live imaging, and the chemical and genetic modulation of a human hillock culture system, we will dissect the molecular mechanisms of hillock differentiation and hillock-mediated injury repair with a focus on the specific role of retinoic acid signaling and the association of hillocks to squamous metaplasia.