A Single-Cell Atlas of the Upper Respiratory Epithelium Reveals Heterogeneity in Cell Types and Patterning Strategies

The upper respiratory tract, organized along the pharyngolaryngeal-to-tracheobronchial axis, is essential for homeostatic functions such as breathing and vocalization. The upper respiratory epithelium is frequently exposed to pollutants and pathogens, making this an area of first-line defense against respiratory injury and infection. The respiratory epithelium is composed of a rich array of specialized cell types, each with unique capabilities in immune defense and injury repair. However, the precise transcriptomic signature and spatial distribution of these cell populations, as well as potential cell subpopulations, have not been well defined. Here, using single cell RNAseq combined with spatial validation, we present a comprehensive atlas of the mouse upper respiratory epithelium. We systematically analyzed our rich RNAseq dataset of the upper respiratory epithelium to reveal 17 cell types, which we further organized into three spatially distinct compartments: the Tmprss11a+ pharyngolaryngeal, the Nkx2-1+ tracheobronchial, and the Dmbt1+ submucosal gland epithelium. We profiled/analyzed the pharyngolaryngeal epithelium, composed of stratified squamous epithelium, and identified distinct regional signatures, including a Keratin gene expression code. In profiling the tracheobronchial epithelium, which is composed of a pseudostratified epithelium-with the exception of the hillock structure-we identified that regional luminal cells, such as club cells and basal cells, show varying gradients of marker expression along the proximal-distal and/or dorsal-ventral axis. Lastly, our analysis of the submucosal gland epithelium, composed of an array of cell types, such as the unique myoepithelial cells, revealed the colorful diversity of between and within cell populations. Our single-cell atlas with spatial validation highlights the distinct transcriptional programs of the upper respiratory epithelium and serves as a valuable resource for future investigations to address how cells behave in homeostasis and pathogenesis. Highlights- Defined three spatially distinct epithelial compartments, Tmprss11a+ pharyngolaryngeal, Nkx2-1+ tracheobronchial, and Dmbt1+ submucosal gland, comprising 17 total cell types - Profiled Keratin gene expression code along proximal-distal and basal-luminal axes and highlighted "stress-induced" Keratins KRT6A and KRT17 at homeostasis - Demarcated expression gradients of Scgb1a1+ and Scgb3a2+ club cells along the proximal-distal axes - Specified submucosal gland cell heterogeneity including Nkx3-1+ mucin-producing cells, with ACTA2+ basal myoepithelial cells exhibiting gene profile for neuroimmune mediated signaling