The alveolar region of the lung develops in a period that spans the late embryonic and early postnatal stages of life. An intricate series of events—including cellular proliferation, surfactant production, and morphogenesis of the alveolar structure—occurs during the transition to air breathing, a process known as alveologenesis. This critical period establishes the spatial arrangement of alveolar epithelium, capillary endothelium, and fibroblasts to generate the gas-exchange niche.

The temporal and spatial alignment of cell compartments and the intercellular signaling that coordinates the development and maturation of the lung alveolus remain poorly characterized. Because of the extensive morphological changes that shape the alveolar niche, it is unclear which subsets of mesenchyme exert mechanical force to remodel alveolar architecture during early postnatal development. Single-cell sequencing and new genetic lineage–tracing tools have helped elucidate the cellular heterogeneity in all three cellular compartments in the lung, with extensive heterogeneity in the lung mesenchyme being of particular note. We sought to integrate these new technologies and tools to assess the cell-cell communication that drives alveolar generation.

We generated a single-cell RNA-sequencing (scRNA-seq) atlas of the developing mouse lung that included epithelial, endothelial, and mesenchymal compartments from time points that span embryonic and postnatal stages. We then analyzed ligand and receptor interactions and identified the alveolar type 1 (AT1) epithelial cell as a hub of ligand expression. The cognate receptors for these ligands were restricted to subsets of developing mesenchymal cells. Mesenchymal progenitors are spatially and transcriptionally segregated into Acta2-, Pdgfrb-, or Wnt2-expressing subsets and are committed to generating distinct fibroblasts in the postnatal lung by embryonic day 15.5 (E15.5). We show with scRNA-seq and lineage tracing that the progenitors for the transient secondary crest myofibroblast (SCMF), which exists only during the early postnatal alveolarization period of lung development, are spatially and transcriptionally aligned with AT1 cell progenitors. In comparison with other alveolar fibroblasts, SCMFs exert significantly more traction force ex vivo, indicating that they are a functionally specialized lineage that can remodel the alveolus. To identify intercellular signaling pathways that regulate cell lineage identity, we examined the single-cell chromatin accessibility and pathway expression (SCAPE) of AT1s and SCMFs. We identified Foxa and Tead transcription factors as upstream regulators of several AT1-derived ligands, including Shh and Wnt ligands. Conversely, SCMFs exhibit open chromatin with predicted Gli1 and Tcf target genes, implicating Shh and Wnt pathways in their development and function. To test these pathways, we generated AT1 cell–specific conditional deletions for Wnt-ligand secretion (Wls) and Shh. Conditional ablation of Shh from AT1 cells results in a loss of SCMF cells and subsequent alveolar simplification in the postnatal lung.

Integrating single-cell genomics and lineage tracing, we identified the spatial and temporal patterning of intercellular signaling pathways that are active during the development and maturation of the distal lung. The AT1 epithelial cell, previously thought to primarily provide gas exchange with capillary endothelium, is also a crucial ligand-expressing node that is required for proper lung development. These observations show that the viability of the AT1 cell is paramount to establish tissue homeostasis during lung …