As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.

中文翻译：

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单细胞缺失分析显示 Tcf7、Spi1、Gata3、Bcl11a、Erg 和 Bcl11b 对 pro-T 细胞发育速度和途径的控制

随着早期 T 细胞前体从多能性转变为 T 谱系定型，它们会改变多种转录因子的表达。目前尚不清楚单个转录因子是否直接控制 T 细胞身份和某些替代命运之间的选择，或者这些因素是否主要影响正常承诺过程中的增殖或存活。在这里，我们揭示了在早期 T 细胞发育的两个阶段删除单个转录因子的影响，使用同步体外分化系统、具有批量索引的单细胞 RNA-seq 和受控基因破坏策略。首先，使用定制的单细胞 CRISPR 破坏方法，我们定义了早期作用转录因子 Bcl11a、Erg、Spi1 (PU.1)、Gata3 和 Tcf7 (TCF1) 在定型前的功能。结果揭示了 T 细胞因子 Tcf7 和 Gata3 与祖细胞因子 Spi1 和 Bcl11a 之间的单个细胞内的动力学拉锯战，对 Erg 具有意想不到的指导作用。其次，我们测试了在承诺过程中转录因子 Bcl11b 的激活如何改变正在进行的细胞程序。在 Bcl11b 表达被阻止的敲除细胞中，细胞没有经历发育停滞，而是随着 T 谱系定型被阻断而遵循另一条路径。一个逐步的、时间依赖性的调节级联开始于立即早期转录因子激活和 E 蛋白抑制，最终导致 Bcl11b 敲除细胞退出 T 细胞通路。最后，从单细胞转录组结果中提取转录因子交叉调控的基因调控网络，