(437) Sox9 and Lef1 regulate the fate and behavior of airway glandular stem cells in response to injury

Background: Cartilaginous airways of larger mammals and the mouse trachea contain at least three well-established stem cell compartments including basal cells (BC) of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Gland progenitors have been previously shown by our lab to contribute to the SAE repair and Lef1 played a critical role in this process (Lynch et al., 2018). A parallel publication showed that Sox9+ gland progenitors also regenerate the SAE after a severe injury (Tata et al., 2018). In this work we provide new insights into how Sox9 and Lef1 regulate processes involved in glandular stem cell recruitment and repair of the airway surface.

Methods: We utilized a variety of conditional genetic mouse models to both lineage trace and/or knockout to study the fate of glandular progenitors and the involvement of Lef1 and Sox9 in regulating their behavior following injury.

Results: Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline as mice transition from juvenile (4 weeks) to adult age (7 weeks). The contribution to the airway surface regeneration by Sox9 glandular progenitors declines ~6 fold when mice are induced at adult age. Some of this difference can be accounted by the ~1.7 fold decline in Sox9-CreER lineage traced ductal progenitors in adult compared to juvenile mice. Sox9-lineage traced glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair and its deletion significantly reduced proliferation ~6 fold following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation in both SAE and SMG progenitors shortly following injury, which is consistent with a proposed role of Sox9 as a Wnt inhibitor in other tissues. Notably, both the surface and gland progenitors failed to proliferate in vitro after Sox9 deletion, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. We frequently observed cell doublets where one of the cells was Ki67+ and the other one was Sox9+, potentially suggesting that an asymmetric cell division event gives rise to an amplifying progenitor cell and a slow-cycling Sox9+ stem cells. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not SAE, progenitors.

Conclusions: We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively (see the graphical abstract for details).

Acknowledgements:

References: Lynch, T. J., Anderson, P. J., Rotti, P. G., Tyler, S. R., Crooke, A. K., Choi, S. H., ... & Engelhardt, J. F. (2018). Submucosal gland myoepithelial cells are reserve stem cells that can regenerate mouse tracheal epithelium. Cell stem cell, 22(5), 653-667.



Tata, A., Kobayashi, Y., Chow, R. D., Tran, J., Desai, A., Massri, A. J., ... & Tata, P. R. (2018). Myoepithelial cells of submucosal glands can function as reserve stem cells to regenerate airways after injury. Cell stem cell, 22(5), 668-683.