A hierarchy of migratory keratinocytes maintains the tympanic membrane

Abstract

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. 1N).The same approaches were utilized to define and locate the cell populations in the fibrous/mucosal fraction, where 12 clusters were discovered. Among these are multiple Vimentin+ (Vim) mesenchymal populations, including: endothelium (Pecam1+), smooth muscle (Acta2+), and Schwann cells (Mbp+) (Figure S3). Four additional clusters of mesenchymal cells were identified, numbered 0, 2, 3, and 5. Inspection of the differentially expressed genes revealed that cluster 0 is Gpx3+, and clusters 2, 3, and 5 are Igfbp3+ (Figure S4A-B). ISH confirmed that Gpx3 and Igfbp3 do indeed mark distinct populations of fibroblasts (Figure S4C). The Gpx3+ cells appear to reside in proximity to bony elements-specifically, in the annular region and near the malleus. The Igfbp3+ cells are more diffusely spread throughout the fibrous layer of the TM.Lastly, Sox2 is a known marker of mucosa, and is expressed in both non-ciliated (clusters 4 and 11)

Developmental aspects of the tympanic membrane: Shedding light on function and disease

“…BrdU‐labeled cells were observed in the same areas as the integrins; along the handle of malleus and the annulus (Kakoi & Anniko, ). This finding has recently been confirmed using another thymidine analogue, EdU, in a single injection and chase experiment as well as a continuous label and chase experiment which additionally established a turnover time of 21 days for TM keratinocytes in mice (Frumm et al, ).…”

“…The middle lamina propria layer also houses the vessels and nerves that supply the TM. Recent single‐cell RNA sequencing data, looking at the cellular makeup of the layers of the TM, supports this established histological knowledge of TM structure, showing the predictable presence of mesenchymal, endothelial, smooth muscle, and Schwann cell clusters in the middle and inner layers of the TM, as well as clusters of keratinocytes at varying stages of differentiation in the outer epidermal layer (Frumm et al, ). This three‐ply structure is maintained in both the pars flaccida and pars tensa with important differences in the cellular makeup of the middle lamina propria layer.…”

“…These are the anterior and posterior malleolar folds that mark the division between the pars tensa and pars flaccida (Figure c). Interestingly, this area has recently been proposed to house a key stem cell population of keratinocytes with long‐term renewal capability (Frumm et al, ).…”

“…Cell dynamics in the outer epidermal layer of the tympanic membrane. (a) Location and movement of stem/progenitor cells based on lineage tracing experiments and proliferation assays (Frumm et al, ; Knutsson, von Unge, & Rask‐Andersen, ). (b) Migration patterns of keratinocytes based on ink dye labeling experiments in rodents and humans (Jackler, Santa Maria, Varsak, Nguyen, & Blevins, ; Michaels & Soucek, ).…”

“…A similar migration pattern was observed in Ki67‐CreERT2; mTmG TMs. Using confetti reporter mice, with the above cre lines in vivo, clonal units of keratinocytes were evident in the TM epidermis, streaking downwards from the malleolar folds over a 3‐month period (Frumm et al, ). This is in contrast to the radial movement seen in historic dye studies (Alberti, ; R. K. Jackler et al, ; L. Michaels & Soucek, ; Figure b), which may have been confounded by inadvertent injury with the placement of dye or may have been reflecting migration from committed progenitor zones, such as the manubrium.…”