CXCL12 defines lung endothelial heterogeneity and promotes distal vascular growth

In adults, there is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury, however, molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to a large number of lung diseases in children, indicating a need to uncover fetal programs that promote pulmonary vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation, gene ontology analysis, and spatial transcriptomics allowed us to segregate the developing artery into spatially and functionally distinct subpopulations. In addition, expression of Cxcl12 suggests a morphogen gradient from arteries to capillaries, suggesting directed cell migration for pulmonary vascular development. Disruption of this gradient led to abnormal branching and pulmonary vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development. Single Cell RNAseq of dissociated mouse lungs at ages E13.5, E15.5, E18.5, and P8 then sorted for Cxcl12-DsRed+ cells (DsRed+/CD31+/EpCAM-/CD45-). Further, Cxcl12 homozygous null (Cxcl12DsRed/DsRed) embryos at E18.5 were also analyzed and compared to Cxcl12 heterozygous (Cxcl12DsRed/+) E18.5 controls.