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.

中文翻译：

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脊索动物细胞命运分歧的单细胞分析


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主要机制。模型脊索动物海鞘中的大多数卵裂球都局限于 64 细胞阶段和原肠胚中期阶段之间的单一主导命运。深度刻板且特征明确的 Ciona 胚胎细胞谱系允许在新建立的细胞类型与兄弟细胞状态分化的早期对其进行转录组分析，而无需分析不太同步的细胞群时所需的伪时间推断。这是第一个使用液滴 scRNAseq 进行大量分析细胞的海鞘研究，最早可追溯到 64 细胞阶段，此时主要谱系（如初级脊索）首先受到命运限制。我们鉴定了 59 种不同的细胞状态，包括 b 系神经谱系的新亚区域和尾尖表皮的早期诱导。我们发现其中 34 种细胞状态直接或间接依赖于 MAPK 介导的信号传导，这对于早期 Ciona 模式形成至关重要。大多数 MAPK 依赖性分叉都通过 MAPK 抑制而失去信号诱导的细胞命运，但后内胚层的独特之处在于转化为表达内胚层和肌肉的一些但不是全部标记物的新状态。新分叉的兄弟细胞类型之间的不同基因表达主要是由诱导细胞类型的上调引起的。 Ets 家族转录因子 Elk1/3/4 在几乎所有假定的直接诱导中都独特地上调。 Elk1/3/4 上调与 Ets 转录因子结合位点富集分析相结合，可以推断出哪些分叉直接或间接受 MAPK 信号控制。 我们详细检查了脊索诱导，发现 Zic/Ets 介导的调节状态和 Brachyury/FoxA 介导的调节状态之间的转变出人意料地晚了。这支持了细胞命运规范的“宽沙漏”模型，其中许多早期组织特异性基因通过同一组转录输入与关键组织特异性转录调节因子并行诱导。