P96. Notch signaling regulates epithelial Clara/cilia fate selection and mesenchymal arterial SMC determination during lung organogenesis

Abstract

Lung development is the result of complex interactions between three tissues: epithelium, mesenchyme, and endothelium. Despite suggestions that Notch signaling plays an important role in this process, the precise function for Notch signaling and the cells in which it is required remains a mystery. In this study, we have attempted to decipher all roles for Notch signaling in developing lung epithelium and mesenchyme respectively using genetic technology. We have created the N1IP:CRE, R26R mice that allow us to map the lineages of cells experiencing at least one round of Notch1 activation and discovered involvements of Notch signaling in the Clara, vascular smooth muscle and endothelial cell fate. We conducted conditional deletion of Notch signaling in lung epithelium and mesenchyme respectively using tissue-specific Cre-expressing mouse lines. In the epithelium, RBPj, a core component of Notch signaling, deletion massively expanded ciliated cell population at the expense of Clara cells. Analysis of Notch1 activation patterns in developing lung epithelia identified N1ICD-containing cells interspaced with Foxj1-positive prospective ciliated cells. These data indicates that Notch signaling determines the Clara cell fate by suppressing a default ciliated cell fate through Notch-mediated lateral inhibition. Stepwise removal of Notch receptors further revealed that Notch2 plays an important role in the cell fate determination and the quantity of Notch receptors reflect CC10 gene, a Clara cell marker, expression level. In the mesenchyme, Notch signaling was required for the recruitment/specification of vascular smooth muscle cells (vSMCs), but not bronchial smooth muscle cells (bSMCs), from a pool of mesenchymal progenitors. We find that RBPj-deficient mesenchymal cells fail to express PDGFR-β that is an indispensable component for vSMC development and known as a direct target of Notch signaling. Collectively, we propose that the primary roles for Notch signaling in lung development are Clara/ciliated cell selection and vasculature formation.