Single-cell RNA-sequencing of angioblasts and endothelial cells from E9.5-E11.5 mouse embryos

Background: The formation of the lymphatic system has been a subject of controversy for over a century. Although the accepted view is that lymphatic endothelial cells originate exclusively from veins, there have been reports of non-venous derived lymphatics in multiple tissues. Methods: We performed single-cell RNA sequencing analyses on ~20000 individual cells isolated from Pax3Cre/+;Rosa26tdTomato mouse embryos at E9.5, E10.5 and E11.5, the temporal window in which lymphatic endothelium initially emerges. Results: These analyses revealed the existence of populations of lymphangioblasts which arise from paraxial mesoderm and directly incorporate into the lymph sacs. Our findings support the notion that lymphatic endothelial cells also have non-venous origins and explain the discrepancies between previous studies. Endothelial progenitors and endothelial cells were isolated from Pax3Cre/+;Rosa26tdTomato embryos at ~E9.5, ~E10.5 and ~E11.5, to define transcriptional differences between paraxial mesoderm-derived and and non-paraxial mesoderm-derived endothelium. Single cell gene expression was profiled using the 10x Genomics Chromium Next GEM Single Cell 3' GEM, Library and Gel Bead Kit v3.1.背景：淋巴系统的形成机制百余年来始终存在学术争议。当前学界公认的观点认为，淋巴内皮细胞（lymphatic endothelial cells）仅起源于静脉，但已有多项研究在多种组织中报道了非静脉来源的淋巴管。 方法：本研究对采集自E9.5、E10.5及E11.5阶段Pax3Cre/+;Rosa26tdTomato小鼠胚胎的约20000个单个细胞开展了单细胞RNA测序（single-cell RNA sequencing）分析，上述阶段正是淋巴内皮细胞初始出现的时间窗口。 结果：本研究的测序分析揭示了一群起源于轴旁中胚层（paraxial mesoderm）的淋巴成管祖细胞（lymphangioblasts），这类细胞可直接整合至淋巴囊之中。本研究结果支持"淋巴内皮细胞亦存在非静脉来源"这一观点，并解释了既往多项研究之间的结论分歧。后续实验中，研究人员分离了约E9.5、E10.5及E11.5阶段Pax3Cre/+;Rosa26tdTomato胚胎中的内皮祖细胞与内皮细胞，以明确轴旁中胚层来源与非轴旁中胚层来源内皮细胞之间的转录组差异。本研究采用10x Genomics Chromium Next GEM Single Cell 3' GEM、Library and Gel Bead Kit v3.1试剂盒完成了单细胞基因表达谱的分析。