Preparing for the first breath at single cell level [whole lung time course]

The respiratory system undergoes remarkable structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify dynamic changes in gene expression in the diverse pulmonary cells at birth, we performed Drop-seq based massive parallel single-cell RNA sequencing. An iterative cell type identification strategy was used to unbiasedly identify the heterogeneity of murine pulmonary cell types on postnatal day 1. Distinct populations of epithelial, endothelial, mesenchymal, and immune cells were identified, each containing distinct subpopulations. Cell type predictions and signature genes identified using Drop-seq were cross-validated using an independent single cell isolation platform. Temporal changes in RNA expression patterns were compared before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, demonstrating activation of UPR signaling during perinatal adaptation of the lung. Present data provide the first single cell view of the adaptation to air breathing after birth. All data from the present study are freely accessed at https://research.cchmc.org/pbge/lunggens/SCLAB.html. Embryos and mice for this study were collected from timed pregnant mice. Whole lungs were surgically dissected at embryonic (E) days 16.5, 18.5 and postnatal days (PND) 1, 3, 7, 14, and 28呼吸系统会发生显著的结构、生化与功能改变，以适应出生后空气呼吸的生理需求。为解析出生后不同肺组织细胞中基因表达的动态变化，我们开展了基于Drop-seq（Drop-seq）的大规模并行单细胞RNA测序。我们采用迭代式细胞类型识别策略，无偏地鉴定了出生后第1天小鼠肺组织细胞类型的异质性。本研究鉴定得到了上皮细胞、内皮细胞、间充质细胞与免疫细胞等不同细胞群，每类细胞均包含独特的亚群。本研究通过独立的单细胞分离平台，对Drop-seq鉴定得到的细胞类型预测结果与特征基因进行了交叉验证。通过对比出生前后RNA表达模式的时序变化，我们筛选得到在特定肺组织细胞中选择性激活的信号通路，证实了肺脏围产期适应过程中未折叠蛋白反应（UPR）信号通路的激活。本研究数据首次从单细胞层面揭示了出生后机体对空气呼吸的适应机制。本研究的全部数据可在以下网址免费获取：https://research.cchmc.org/pbge/lunggens/SCLAB.html。本研究的胚胎与小鼠均取自定时交配的孕鼠，研究人员分别在胚胎期（E）16.5天、18.5天以及出生后天数（PND）1、3、7、14和28天，通过手术分离获取完整肺组织。