Early lineage commitment defines alveolar epithelial ontogeny in the lung

During the step-wise specification and differentiation of tissue specific multipotent progenitor cells, lineage-specific transcriptional networks are either activated or repressed to orchestrate progenitor cell commitment. The gas exchange niche in the lung contains two major epithelial cell types, alveolar type 1 (AT1) and type 2 (AT2) cells, and the timing of lineage commitment of these cells is critical for correct formation of this niche and postnatal survival. To define the ontogeny of alveolar cell fate in the lung, we used lineage tracing studies combined with spatially specific mRNA transcript and protein expression combined with single cell RNA-seq analysis. These studies reveal that commitment to alveolar epithelial cell fate occurs far earlier than previously appreciated, concomitant with the proximal-distal specification of epithelial progenitors and branching morphogenesis. Using a novel dual lineage tracing system, we show that a small population of alveolar cells express markers of both AT1 and AT2 cells, whose fate is ultimately restricted to a single lineage. However, these bi-transcriptional cells generate only a minor portion of the mature alveolar epithelium. These data reveal a new paradigm of organ formation where early lineage commitment occurs during the nascent stages of development coincident with broad tissue patterning processes including axial patterning of the endoderm and branching morphogenesis. Lungs were harvested from a e15.5 and e17.5 Nkx2-1-GFP embryos and all were combined from a single litter. Dead cells were negatively selected using DAPI and positively selected by expression of GFP. Cells were loaded on to a GemCode instrument (10X Genomics) to produce single-cell bar-coded droplets using 10X Single Cell 3' v2 chemistry. Libraries generated were sequenced across at least one lane on the Illumina HiSeq2500 instrument with the HiSeq Rapid SBS kit.在组织特异性多能祖细胞的逐步特化与分化进程中，谱系特异性转录调控网络会被激活或抑制，以调控祖细胞的谱系定型。肺内的气体交换微环境（gas exchange niche）包含两种主要上皮细胞类型：肺泡1型（alveolar type 1, AT1）与肺泡2型（alveolar type 2, AT2）细胞，此类细胞的谱系定型时序对于该微环境的正确形成及出生后存活至关重要。 为阐明肺内肺泡细胞命运的个体发生历程，本研究结合谱系示踪（lineage tracing）技术、空间特异性mRNA转录本与蛋白质表达分析，以及单细胞RNA测序（single cell RNA-seq）技术开展研究。上述研究揭示，肺泡上皮细胞的谱系定型发生时间远早于此前认知，且与上皮祖细胞的近-远轴特化及分支形态发生过程同步进行。 本研究利用一种新型双谱系示踪系统，发现少量肺泡细胞可同时表达AT1与AT2细胞的标志物，此类细胞的命运最终会被限定为单一谱系；但这类双转录表型细胞仅占成熟肺泡上皮的极小部分。上述数据揭示了器官形成的全新范式：早期谱系定型发生于发育的初始阶段，且与包括内胚层轴模式建成及分支形态发生在内的广泛组织模式建成进程同步进行。 本研究收集了胚胎日龄15.5天（E15.5）与17.5天（E17.5）的Nkx2-1-绿色荧光蛋白（Nkx2-1-GFP）小鼠胚胎肺组织，所有样本均取自同窝胚胎。采用4',6-二脒基-2-苯基吲哚（DAPI）进行死细胞阴性分选，并通过绿色荧光蛋白（GFP）表达进行阳性分选。将分选后的细胞上样至GemCode仪器（10X Genomics），利用10X单细胞3'端v2建库试剂盒制备带有单细胞条形码标记的微滴。制备得到的文库在Illumina HiSeq2500测序仪的至少一个测序泳道上，采用HiSeq快速SBS测序试剂盒完成测序。