Transitioning lung for postnatal life
The lung is a complex organ composed of multiple cell types, and its alveolus serves as the functional unit of gas exchange. The alveolar type 1 cell (AT1) serves as an active signaling hub in the developing and postnatal mouse and human lung. Zepp et al. generated a comprehensive single-cell atlas of the developing murine lung and identified cell differentiation and cell-to-cell communication as the lung transitions to air breathing. The AT1 cells spatially aligned with stromal progenitors and formed a signaling hub that preferentially communicated with a transient, force-exerting, myofibroblast through signaling factors including Shh and Wnts to actively remodel the alveolus after the transition to air breathing.
Science, this issue p. eabc3172
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 development. Our imaging and single-cell biophysical measurement assays show that AT1-adjacent mesenchymal progenitors occupy anatomically discrete regions and are functionally specialized to mold the intricate architecture of the alveolus. These findings underscore the transcriptional and functional heterogeneity in distinct lung fibroblast lineages.
A lung single-cell developmental atlas reveals an enriched signature of ligand expression in AT1 epithelial cells. Chromatin accessibility and whole-mount imaging identify the transcriptional and spatial alignment of AT1 and SCMF progenitors in the developing lung. AT1-derived Shh is required for specification and outgrowth of the force-exerting SCMF.
The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remains poorly understood. Applying a multimodal approach, we identified alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting myofibroblasts in a spatial and temporal manner. Through single-cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration.