Genetic determinants of epithelial differentiation in the mouse and human lungs

Abstract: Several airway epithelial cell types and subpopulations have been defined, using high throughput single-cell mRNA profiling. However, the spectrum and topology of cell populations within the secretory cell lineage has not been explored. The subject of this thesis is the investigation of diverse secretory cell states, the identification of their progenitors and the exploration of genetic mechanisms establishing distinct secretory cell states in the airways.Hedgehog (HH) signaling is important for airway development. The paracrine function of epithelial-derived HH expression is to pattern the adjacent developing mesoderm and give rise to the future mesenchyme. In turn, patterning of the mesenchyme facilitates proper airway epithelial differentiation. However, a possible autocrine role of HH signaling on the developing epithelium and whether or not it participates in airway epithelial differentiation has remained unexplored. We utilized knockout mouse models and an in vitro culture setup of human bronchiolar epithelial cells (HBECs), to investigate the autocrine function of HH signaling, in mice and humans. Epithelial specific inactivation of the Smoothened (Smo) effector in the developing trachea, rendered epithelial cells unresponsive to HH signaling. Tracheal epithelial cells, deficient for Smo, showed reduced proliferation of epithelial cells and their differentiation towards ciliated and secretory cell types. The observed phenotype was milder than the one caused by epithelial inactivation of the ligand sonic hedgehog (Shh), presumably due to changes in the mesenchyme that signals in a paracrine fashion to regulate epithelial differentiation. We found that autocrine function of HH signaling in tracheal epithelial cells promotes secretory and ciliated cell differentiation from epithelial progenitor cells. Pharmacological in vitro inactivation of Smo activity in HBECs shows a conserved function of HH signaling in airway development in mammals. So, our data conclude that Smo activity in tracheal epithelial cell controls the proliferation of epithelial progenitors and their differentiation in cell-autonomous manner.Secretory cells are the bulk cell type of the airway epithelium. To further investigate the potential heterogeneity within the secretory cell lineage and the mechanisms that control the balance between the secretory subpopulations, we combined single cell transcriptomic profiling with a multiplex hybridization approach. We found opposing gradients of differentiation, along the proximodistal axis of the adult lung epithelium. Within these gradient programs, we defined two distinct secretory cell states S1 and S2, each expressing a unique set of mature markers. A third, secretory state is defined by the low levels of expression of both S1 and S2 markers, suggesting that it represents an intermediate default state. The three secretory states show distinct localization along the proximal-distal airway axis. Using transgenic mice, we inactivated fibroblast growth factor receptor (FGFR) signaling shortly after birth, specifically in all secretory cells. We found that FGFR deficient cells reduced the levels of distally expressed markers, including epithelial type 2 (AEC2) -related genes and upregulated AEC1-related genes. This suggests FGFR activity promotes proper distalization of the airway epithelium and is maybe required for the function of distal bronchiolar secretory cells in homeostasis and upon injury.