Single-cell RNA-seq of tailbuds from control, Pbx1/Pbx2 compound and Pbx1/Pbx2 double-knockout E8.5/E9.0 embryos

In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). NMPs reside in the caudal lateral epiblast and tailbud, from where they sustain axial elongation. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm, single-cell RNA sequencing (scRNA-seq) was performed on cells isolated from the tailbuds of control, Pbx1/Pbx2 compound (Pbx1/2-com) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) embryos at embryonic days 8.5 and 9.0. Single cells from at least three embryos per genotype and stage were FACS-sorted into 384-well capture plates, and scRNA-seq was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). As a spike-in internal control for batch-effect correction, 71 EpiSCs cultivated in vitro were sorted into each plate, together with 311 cells from embryonic tailbuds. Two wells were left empty in each plate, as a no-cell control during data analysis.在脊椎动物中，体轴延伸由双能神经中胚层祖细胞（bipotent neuromesodermal progenitors, NMPs）驱动，这类细胞可同时支持脊髓与体节中胚层（paraxial mesoderm, PM）的发育。NMPs定位于尾侧外侧上胚层与尾芽中，并通过此处维持体轴延伸过程。长期以来，HOX转录因子被认为与体轴延伸密切相关，其依次激活在体节中胚层发育的时序调控中发挥核心作用。PBX1与PBX2是必需的前部HOX辅因子，因此它们是调控不同HOX因子特异性应答的关键候选靶点。为精准解析PBX蛋白在体节前中胚层发育中的作用，研究人员对胚胎发育第8.5天和第9.0天的对照组、Pbx1/Pbx2复合突变（Pbx1/2-com）及Pbx1/Pbx2双基因敲除（Pbx1/2-DKO）胚胎的尾芽分离细胞开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。每个基因型与发育阶段的细胞均取自至少3枚胚胎，经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）后接种至384孔捕获板，随后采用MARS-seq技术完成测序（Jaitin D.A.等，《科学》，343卷，776-779页，2014年）。为实现批次效应校正，研究人员在每块板中加入71株体外培养的上胚层干细胞（epiblast stem cells, EpiSCs）作为外源内参，同时纳入311枚胚胎尾芽来源的细胞。每块板预留2个空孔，作为数据分析阶段的无细胞对照。