Release of Notch signaling coordinated by inflammation confers cross-compartment differentiation plasticity during alveolar regeneration.

While the acquisition of cellular plasticity in adult stem cells is essential for rapid restoration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Here, we reveal that Notch signaling is a pivotal determinant conferring cross-compartment differentiation plasticity of airway secretory cells. Temporal Notch inhibition in secretory cells is required for alveolar differentiation upon injury, which is mediated by IL-1β-dependent modulation of Jag1/2 expression in ciliated cells. We also identify Fosl2/Fra2 as an essential transcription factor responsible for the regeneration of secretory cell-derived alveolar type 2 (sAT2) cells retaining distinct genetic/epigenetic signatures. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate that Notch signaling acts as a functional rheostat for fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration. For lineage-labeled cells from Red2-NotchN1ICD mice, YFP+CD45-CD31-EpCAM+ or RFP+CD45-CD31-EpCAM+ cells were sorted at day 28 post bleomycin injury (4 mice were pooled for each experiment). For non-lineage-labeled cells isolated from Red2-NotchN1ICD mice in parallel with experiment of lineage-labeled cells, we combined the cells of EpCAM+RFP-RFP- and EpCAM- population with a ratio of 1:1, respectively. The resulting cell suspension (~110,000 cells each) were submitted as separate samples to be barcoded for the droplet-encapsulation single-cell RNA-seq experiments using the Chromium Controller (10X Genomics). Single cell cDNA synthesis, amplification, and sequencing libraries preparation were performed using the Single Cell 3' Reagent Kit as per the 10x Genomics protocol. Libraries were multiplexed so that 2 libraries were sequenced per single lane of HiSeq 4000 using the following parameters: Read1: 27 cycles, i7: 9 cycles, i5: 0 cycles; Read2: 100 cycles to generate 150bp paired end reads.