Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development
Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves Molecular Drivers of Early Human Endoderm Development
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DOI：
10.1016/j.celrep.2019.03.076
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发表时间：
2019-04-16
期刊：
影响因子：
8.8
通讯作者：
Maehr, Rene
中科院分区：
文献类型：
--
作者：
Genga, Ryan M. J.;Kernfeld, Eric M.;Maehr, Rene
脊椎动物的研究已经概述了定形内胚层（END）发育的保守分子控制。然而，最近的研究也表明，人类END调控的关键分子方面甚至与啮齿动物不同。人胚胎干细胞（ESC）向END的分化为研究正常和有缺陷的人特异性END发育的分子基础提供了一个易处理的系统。在这里，我们询问了在胚胎干细胞分化为END过程中染色质可及性的动态变化，预测了可能驱动这种细胞命运转变的DNA结合蛋白。然后，我们将单细胞RNA-seq与平行CRISPR扰动相结合，以全面定义END发展中这些因素的功能丧失表型。在几个候选人之后，我们揭示了TGF β信号传导介质的分化轨迹中的明显损伤，并揭示了FOXA 2转录因子在引发人前肠和肝END特异性的人END能力中的作用。总之，这项单细胞功能基因组学研究为人类END的发展提供了高分辨率的见解。
Studies in vertebrates have outlined conserved molecular control of definitive endoderm(END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFb signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.