Abstract Rationale Notch3-positive mural cells, including pericytes and vascular smooth muscle, ensheath blood vessels and are necessary for vascular development, stabilization, and contraction. Though functionally relatively distinct, the transcriptomic diversity remains incompletely understood. Methods To identify phenotypically specific genes, we performed single-cell RNA sequencing on FACS-isolated Notch3-positive mouse lung cells. To confirm the presence of cell subtypes, we used RNA-scope in situ hybridization. Results We identified six characteristic cell subtypes in the developing mouse, including one pericyte cluster, a proliferating cluster, two vascular smooth muscle clusters (contractile and proliferating), a novel Steap4+ cluster, and a novel Dlk1+ cluster, present during the saccular stage of early lung development, postnatal day (P) 3, and disappearing by the onset of early alveolarization, P7 in the murine lung. Isolating Notch3/NOTCH3+ cells in silico in developmental mouse, human infant, and adult human datasets demonstrated the presence of a Steap4+ mural cell in multiple species. RNA-scope in situ hybridization confirmed the presence of both subtypes in developmental mouse lung, with Dlk1+ cells primarily located near intermediate vessels smaller than 50 μm in diameter. Dlk1 is a developmentally enriched, non-canonical NOTCH ligand that tunes lineage commitment and vascular remodeling to either promote or restrain angiogenesis depending on context. Even though only a small subset of lung mural cells contains STEAP4, a multi-tissue Tabula Sapiens analysis showed that the combination of STEAP4 and FABP4 was broadly expressed by mural cells in other tissues, including the heart, liver, and pancreas, among others. Steap4 is a metalloreductase involved in iron and copper homeostasis and is a known responder to inflammatory stress, while the fatty acid-binding protein Fabp4 is an intracellular lipid chaperone crucial for fatty acid metabolism in adipocytes and macrophages. Conclusion These findings reveal a previously undescribed diversity of mural cells in the lung with yet undetermined functional and developmental significance. We speculate that the specialized STEAP4+ pericytes may play a role in the response to injury and repair, while DLK1+ pericytes are critical to lung development. This abstract is funded by: NHLBI, Karam Family Foundation
Forst et al. (Fri,) studied this question.