Single-cell analysis of cell fate bifurcation in the chordate Ciona. Single-cell analysis of cell fate bifurcation in the chordate Ciona

Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly-established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a ‘broad-hourglass’ model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs. Overall design: Whole-embryo single-cell RNA sequencing of Ciona embryos

不同细胞类型之间的诱导信号相互作用，是胚胎细胞命运进一步多样化的核心调控机制。在模式脊索动物强健海鞘（Ciona robusta）中，绝大多数卵裂球会在64细胞期至原肠中期阶段，被限定为单一主要细胞命运。该物种的胚胎细胞谱系具有高度刻板性且已被充分解析，使得我们能够在新生细胞类型刚从姊妹细胞状态分化的极早期，开展转录组分析——无需像分析同步性较低的细胞群那样，进行拟时间（pseudotime）推断。本研究是首个在64细胞期就利用液滴单细胞RNA测序（droplet scRNAseq）分析大量细胞的被囊动物研究，该时期正是原脊索等主要谱系首次确立细胞命运限定的关键节点。我们共鉴定出59种不同的细胞状态，其中包含b系神经谱系的全新亚区域，以及尾尖表皮的早期诱导细胞亚型。我们发现，其中34种细胞状态的形成直接或间接依赖于对强健海鞘早期模式建成至关重要的丝裂原活化蛋白激酶（MAPK）介导的信号通路。多数依赖MAPK的细胞命运分支呈现渠化特征：当MAPK被抑制时，信号诱导的细胞命运会完全丢失；但后内胚层是个特例，它会被转化为一种全新的细胞状态，同时表达内胚层与肌肉细胞的部分而非全部标记基因。新分化的姊妹细胞类型间的差异基因表达，主要表现为诱导产生的细胞类型发生基因上调。ETS家族转录因子（Ets family transcription factor）Elk1/3/4几乎在所有推测的直接诱导事件中，均呈现特异性上调表达。结合Elk1/3/4的上调表达特征与ETS转录因子结合位点富集分析结果，我们可以推断出哪些细胞命运分支直接受MAPK信号调控，哪些则为间接调控。我们对脊索诱导过程展开详细分析，发现从ZIC/ETS介导的调控状态，向短尾蛋白（Brachyury）/FOXA（FoxA）介导的调控状态的转变进程意外延迟。该结果支持细胞命运特化的‘宽沙漏模型’：即大量早期组织特异性基因与关键组织特异性转录调控因子，可通过同一套转录输入信号被同步诱导表达。实验整体设计：对强健海鞘胚胎开展全胚胎单细胞RNA测序。