Single-cell RNA-seq of mouse embryos E4.5 to E7.5. First batch.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers. Useful links: Parsed data: (ftp://ftp.ebi.ac.uk/pub/databases/scnmt_gastrulation) Github repository: (https://github.com/rargelaguet/scnmt_gastrulation) Overall design: 758 single cells isolated from mouse embryos at E4.5, E5.5, E6.5 and E7.5 and processed using scNMT-seq (Clark et al 2018) NOTE: The GSE133725 Series supplementary file, embryo_cell_counts_matrix_030719.tsv, contains total 2971 processe data columns for both GSE121650 (758 cells) and GSE133725 (2213 cells) samples.

原肠胚形成（gastrulation）过程中三胚层的构建，是脊椎动物躯体蓝图建立过程中的关键步骤。近期采用单细胞RNA测序（single cell RNA-sequencing）的相关研究，已鉴定出与胚层特化相关的主要转录组变化。这些变化伴随有全局表观遗传重编程（epigenetic reprogramming），但表观基因组（epigenome）在调控早期细胞命运抉择中的作用仍未明确，不同表观遗传层级间的协调机制亦尚未阐明。
本研究首次报道了小鼠胚胎多能性退出（exit from pluripotency）及原肠胚启动阶段，染色质开放程度（chromatin accessibility）、DNA甲基化（DNA methylation）与RNA表达（RNA expression）的单细胞三重组学（single cell triple-omics）图谱。本研究发现不同分子层级间存在动态关联，并找到了表观遗传调控存在不同模式的相关证据。细胞初始的多能性退出，与全局抑制性表观遗传景观的建立相契合；随后在原肠胚形成过程中，逐步出现局部的谱系特异性表观遗传模式。值得注意的是，已定向为中胚层（mesoderm）与内胚层（endoderm）的细胞会发生广泛的协同表观遗传重排，这一过程由增强子标记（enhancer marks）区域的甲基化丢失，以及伴随而来的染色质开放程度升高所驱动。与之形成鲜明对比的是，外胚层细胞的表观遗传景观在早期上胚层（epiblast）阶段便已奠定。因此，在原肠胚形成过程中的显性细胞命运决定发生之前，与各胚层相关的调控元件（regulatory elements）要么已通过表观遗传预置（epigenetically primed），要么已完成表观遗传重塑（epigenetically remodelled），这为三胚层的层级式出现提供了分子层面的逻辑基础。
相关资源链接：解析后数据：(ftp://ftp.ebi.ac.uk/pub/databases/scnmt_gastrulation) Github仓库：(https://github.com/rargelaguet/scnmt_gastrulation)
实验设计概览：从E4.5、E5.5、E6.5及E7.5阶段的小鼠胚胎中分离得到758个单细胞，采用scNMT-seq技术进行测序处理（Clark等人，2018年）
备注：GSE133725系列的补充文件embryo_cell_counts_matrix_030719.tsv，共包含2971个处理后的数据列，对应GSE121650（758个细胞）与GSE133725（2213个细胞）两个数据集的样本。