The Hillock: A Newly Discovered Regenerative Epithelial Structure in the Airway Epithelium

Abstract

In 2018, the Rajagopal lab identified the airway hillock, a novel murine airway epithelial structure of unknown function. Hillocks have since been reported in human airways, suggesting that they are an evolutionary conserved structure. Most of the airway is covered by pseudostratified epithelium, composed primarily of basal cells, secretory club cells, and ciliated cells. The hillock is distinguished from this epithelium in three major ways: (1) the presence of stratified-appearing layers of flat KRT13+ cells which sit atop hillock basal cells that are KRT14+ (2) the lack of luminal ciliated cells, and (3) the enhanced replication of the basal cells. Using Cre-ER based lineage tracing of the hillock reporter gene Krt13 combined with immunostaining, we found that hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Indeed, using a basal stem cell specific lineage reporter driver (P63Cre-ER), we discovered that the underlying hillock basal stem cells continually replenish overlying squamous barrier cells. In terms of their previously unknown functional role, we found that hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Indeed, using mosaic reporter mice, we found that after naphthalene injury, hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway and eventually regenerate normal airway epithelium with each of its six component cell types. Thus, hillocks are a specialized structure that are a injury-resistant reservoirs of plastic stem cells to regenerate the epithelium post-injury. Additionally, we investigated the regulation of these stem cells in both in vivo and in vitro contexts. Using in vitro air-liquid interface (ALI) culture systems where we can grow airway epithelium from isolated basal stem cells, we found that hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signaling inhibition, a known cause of squamous metaplasia. Indeed, using the in vivo squamous metaplasia model of vitamin A deficiency, we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of ‘squamous metaplasia’, which is long thought to be a precursor of lung cancer.