The mesenchyme consists of heterogeneous cell populations that support neighboring structures and are integral to intercellular signaling, but are poorly defined morphologically and molecularly. Leveraging single-cell RNA-sequencing, 3D imaging and lineage tracing, we classify the mouse lung mesenchyme into three proximal–distal axes that are associated with the endothelium, epithelium and interstitium, respectively. From proximal to distal: the vascular axis includes vascular smooth muscle cells and pericytes that transition as arterioles and venules ramify into capillaries; the epithelial axis includes airway smooth muscle cells and two populations of myofibroblasts – ductal myofibroblasts, surrounding alveolar ducts and marked by CDH4, HHIP and LGR6, which persist post-alveologenesis, and alveolar myofibroblasts, surrounding alveoli and marked by high expression of PDGFRA, which undergo developmental apoptosis; and the interstitial axis, residing between the epithelial and vascular trees and sharing the marker MEOX2, includes fibroblasts in the bronchovascular bundle and the alveolar interstitium, which are marked by IL33/DNER/PI16 and Wnt2, respectively. Single-cell imaging reveals a distinct morphology of mesenchymal cell populations. This classification provides a conceptual and experimental framework applicable to other organs.

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