The emergent landscape of the mouse gut endoderm at single-cell resolution

To comprehensively delineate the ontogeny of an organ system, we generated 112,217 single- cell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. We employed graph-based approaches to model differentiating cells for spatio- temporal characterization of developmental trajectories. Our analysis reveals the detailed architecture of the emergence of the first (primitive or extra-embryonic) endodermal population and pluripotent epiblast. We uncover an unappreciated relationship between descendants of these lineages, before the onset of gastrulation, suggesting that mixing of extra-embryonic and embryonic endoderm cells occurs more than once during mammalian development. We map the trajectories of endoderm cells as they acquire embryonic versus extra-embryonic fates, and their spatial convergence within the gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observe the regionalized localization of cells along the forming gut tube, reflecting their extra-embryonic or embryonic origin, and their coordinate patterning into organ-specific territories along the anterior-posterior axis. Total RNA was extracted from bulk tissue and dissociated cells of 13ss (~E8.75) gut tubes, from bulk tissue from anterior, anterior-midgut, midgut-posterior and posterior sections of 13ss gut tubes, as well as from extra-embryonic visceral endoderm and embryonic visceral endoderm of E7.5 embryos (see also table in section: Bulk RNA processing). The Trizol method (Invitrogen) was used for RNA extraction.为全面勾勒某一器官系统的个体发生（ontogeny），我们生成了112,217个单细胞转录组（single-cell transcriptomes），覆盖小鼠胚胎至妊娠中期的所有内胚层（endoderm）群体。我们采用基于图的（graph-based）分析方法对分化细胞进行建模，以实现发育轨迹的时空（spatio-temporal）表征。本研究揭示了首批（原始或胚外）内胚层群体与多能上胚层（pluripotent epiblast）出现的精细架构。我们发现了原肠运动（gastrulation）启动前，这些谱系后代间未被关注的关联，提示哺乳动物发育过程中胚内与胚外内胚层细胞的混合事件不止发生一次。我们绘制了内胚层细胞获取胚胎与胚外命运的轨迹，以及二者在肠道内胚层中的空间趋同现象，揭示其整体转录组特征相似，但仍保留谱系起源相关的遗留特征。我们观察到细胞沿形成中的肠管呈现区域化定位，这一现象反映了其胚外或胚胎起源，并使其沿前后轴协调模式化为器官特异性区域。我们从13体节期（13ss，~E8.75）肠管的整块组织与解离细胞、13体节期肠管的前、前中肠、中肠-后肠及后肠区段的整块组织，以及E7.5胚胎的胚外脏壁内胚层（extra-embryonic visceral endoderm）与胚胎脏壁内胚层（embryonic visceral endoderm）中提取总RNA（详见「批量RNA处理」（Bulk RNA processing）章节附表）。RNA提取采用Trizol法（Invitrogen）。