The genomic, epigenomic and biophysical cues controlling the emergence of the gas exchange niche in the lung

The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remain poorly understood. Applying a multimodal approach, we identify alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting, myofibroblasts in a spatial and temporal manner. Through single cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration. All mouse experiments used CD-1 female mice with the age indicated in the libraries . Single-cell suspensions were prepared from whole mouse lungs. For all libraries, DAPI-negative cells were were sorted by flow cytometry to obtain heterogeneous whole lung sample. Human tissue was derived from normal peripheral lung tissue. Single cell suspension was depleted of CD45-positive cells by bead selection. All of the murine post-natal timepoints also had CD45-positive cells removed from cell sort. Single-cell barcoded droplets were produced using 10X Single Cell 3' v2 chemistry. Libraries generated were sequenced using HiSeq2500 instrument in High-output mode. Reads were aligned and gene level unique molecular identifier (UMI) counts were obtained using the Cell Ranger pipeline.肺肺泡是呼吸系统中负责气体交换的功能单位。在出生后过渡至空气呼吸的过程中，生物物理力被认为会塑造新生的组织微环境（niche）。然而，驱动这一过程的细胞间信号调控机制仍有待深入解析。本研究采用多模态研究策略，鉴定出肺泡1型（alveolar type 1, AT1）上皮细胞作为一类独特的信号枢纽。谱系示踪实验证实，AT1祖细胞会以时空特异性的方式，与具备信号接收能力且可施加生物物理力的肌成纤维细胞形成空间对齐。通过单细胞染色质开放性与通路表达（single cell chromatin accessibility and pathway expression, SCAPE）分析，我们证明AT1特异性配体对于肌成纤维细胞发育及肺泡形成不可或缺。本研究表明，由生物物理力与AT1来源的旁分泌信号介导的细胞命运对齐，驱动了出生后呼吸功能所需的大规模组织重塑。所有小鼠实验均使用CD-1品系雌性小鼠，小鼠周龄信息已在文库说明中标注。我们从完整小鼠肺组织中制备单细胞悬液：对于所有文库，通过流式细胞术分选出DAPI阴性的异质性全肺样本。人类组织取自正常外周肺组织，通过磁珠分选去除CD45阳性细胞；所有小鼠产后时间点的样本也均剔除了CD45阳性细胞。单细胞标记液滴的制备采用10X Single Cell 3' v2 化学试剂。构建完成的文库使用HiSeq2500仪器的高产出模式进行测序。测序读段通过Cell Ranger流程进行比对，并获取基因水平的唯一分子标识符（unique molecular identifier, UMI）计数。