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. Overall design: 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.

在成年个体中，已有越来越多的研究揭示了肺循环的细胞多样性以及损伤后血管修复的调控机制，但目前对于胚胎发育阶段肺血管形态发生与生长的分子及细胞机制仍不甚明晰。尤为重要的是，血管发育缺陷会引发大量儿童肺部疾病，这凸显了解析促进肺血管生长的胎儿发育程序的迫切需求。为解决这一问题，本研究使用了可报告趋化因子配体12（Cxcl12，一种动脉标志性基因）表达的转基因小鼠品系，并对分离得到的Cxcl12-DsRed+内皮细胞进行单细胞RNA测序，以解析肺内皮细胞的细胞异质性。通过整合细胞注释、基因本体（GO）分析与空间转录组学技术，本研究将发育中的动脉划分为空间分布与功能特性均存在差异的亚群。此外，Cxcl12的表达模式提示存在一条由动脉至毛细血管的形态发生素梯度，这表明肺血管发育过程中存在定向细胞迁移现象。破坏该梯度可导致血管分支异常与肺血管发育不全。本研究数据为动脉内皮细胞的功能异质性提供了直接证据，并揭示了肺血管发育所必需的保守信号调控机制。整体实验设计：对不同发育阶段（E13.5、E15.5、E18.5及P8）解离的小鼠肺组织进行处理，分选得到Cxcl12-DsRed+细胞（DsRed+/CD31+/EpCAM-/CD45-）并开展单细胞RNA测序。此外，本研究还对E18.5的Cxcl12纯合敲除（Cxcl12DsRed/DsRed）胚胎进行分析，并与同阶段的Cxcl12杂合（Cxcl12DsRed/+）对照胚胎进行比较。