A hierarchy of migratory keratinocytes maintains the tympanic membrane

Although the conductive function of the tympanic membrane (TM) is critical for hearing, it is unknown how the organ maintains cellular homeostasis. Using a combination of single-cell RNA sequencing, lineage tracing, whole-organ explant, and live-cell imaging, we demonstrate that the stem cells of the TM epidermis reside in a distinct location at the superior portion of the TM and, as progeny migrate inferiorly, Pdgfra+ fibroblasts maintain a niche supporting proliferation of committed progenitors, while keratinocytes distal from the niche differentiate. Thus, the TM has a three dimensional differentiation hierarchy of keratinocytes distinct from that at other epidermal sites. The TM represents a physiological context where, in the absence of injury, keratinocytes both transit through a proliferative committed progenitor state and exhibit directional lateral migration. This work forms a foundation for understanding common disorders of the TM and introduces a new model system for the understanding of keratinocyte biology.