Cellular Dynamics of Tympanic Membrane Homeostasis and Perforation Repair

The tympanic membrane (TM) is a crucial component of the conductive apparatus of the ear. It defines the border between the external auditory canal (EAC) and middle ear, and it transmits sound vibrations gathered in the EAC to the first middle ear ossicle, the malleus. There is much important pathology of the TM, including perforations (both acute and chronic), cholesteatoma, keratosis obturans, tympanosclerosis, and myringitis. Our lack of understanding of the basic biology of the TM has hampered our understanding of and ability to treat these conditions. The primary goal of the work presented herein was to elucidate mechanisms underlying TM maintenance and wound response.To catalog the specific cell populations of the TM, we conducted single-cell RNA sequencing of dissociated murine and human TM tissue, followed by validation of the populations by immunofluorescence and in situ hybridization of whole-mount and sectioned tissue. We demonstrated that the keratinocytes of the pars tensa epidermis are largely undifferentiated, and that what differentiation occurs is not obligately paired with stratification as it is at other epidermal sites. Furthermore, we studied turnover of the tissue with continuous EdU uptake, and determined that the entire epidermal layer proliferates in approximately three weeks under homeostatic conditions, but in under one week following a perforation. Additionally, we utilized two Cre drivers, Ki67-CreERT2 and Krt5-CreERT2, paired with the Confetti reporter allele to characterize the clonal architecture of the TM epidermis. The development of clones qualitatively supports the model of neutral drift dynamics in the stem cell pool. Features of individual clones suggests that cells in the proliferative region of the superior pars tensa contribute to long-term maintenance of the tissue and can thus be termed stem cells (SC), while cells in the proliferative region of the malleus only transiently contribute cells to the tissue and can thus be considered more committed progenitors (CP). We propose a model in which these distinct SC and CP populations are maintained by the continuous migration of keratinocytes over the TM. Lastly, we identify Pdgfra signaling in fibroblasts as a molecular signal required to maintain the proliferative niches of the TM