Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSC-primed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr+Kit+CD44+, PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.

中文翻译：

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单细胞转录和功能分析揭示了胚胎内皮向第一个造血干细胞的进化。

成年人的造血干细胞（HSC）被认为是由妊娠中期胚胎的造血内皮细胞（HEC）产生的。由于具有罕见和短暂的性质，因此从未严格鉴定和准确捕获具有HSC功能的HEC，更不用说它们的真正血管前体了。在这里，我们首先使用高精度的单细胞转录组学，从胚胎第9.5天到E11.0的0.5天间隔，在连续发育阶段公正地检查了相关的EC种群。结果，我们在转录组上鉴定了两个分子不同的动脉EC群体和推定的HSC引发的HEC，其数量在E10.0达到峰值，然后在主动脉-性腺-中肾（AGM）的背主动脉中急剧下降。结合计算预测和体内功能验证，我们通过新构建的Neurl3-EGFP报告基因小鼠模型精确捕获了具有HSC能力的HEC，并通过结合表面标记（Procr + Kit + CD44 +，PK44）进一步实现了富集。令人惊讶的是，在单个HEC的培养物中很少但可靠地观察到内皮-造血双重潜能。值得注意的是，来自E8.0的原始血管EC经历了两步命运选择，成为HSC引发的HEC，即最初的动脉命运选择，然后是血源性命运转换。这一发现解决了以前观察到的一些矛盾。综上所述，对体内HSC启动的HEC规范所依据的内皮细胞进化和分子程序的全面理解将有助于指导体外HSC生产的未来研究。