The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors [ChIP-Seq]. The T-box Transcription Factor Eomesodermin Governs Hemogenic Competence of Yolk Sac Mesodermal Progenitors [ChIP-Seq]

Extra-embryonic mesoderm (ExM), the earliest cells that traverse through the primitive streak, give rise to the endothelium as well as hematopoietic progenitors in the developing yolk-sac (YS). How a specific subset of ExM becomes committed to a hematopoietic fate remains unclear. Here we report that Eomesodermin (Eomes), a T-box transcription factor, is transiently expressed in ExM progenitors that generate virtually all YS hematopoietic and endothelial cells. Using an embryonic stem cell (ESC) differentiation system, we find that Eomes activity is essential for the production of primitive erythrocytes and for the normal development of Runx1+ HE that generates the definitive hematopoietic progenitors. RNA-Seq and ATAC-Seq experiments reveal that Eomes governs the accessibility of numerous hematopoietic enhancers that SCL normally utilizes to specify primitive erythrocytes and HE in Flk-1hi/PdgfRa- hematovascular mesoderm. ChIP-seq experiments suggest that Eomes coordinates the development of hemogenic competent mesoderm in the context of Activin/Nodal and Tead-Yap signaling. Finally, single-cell- RNA-seq (scRNAseq) shows that in the absence of Eomes function diversion towards an endothelial rather than hematopoietic fate occurs after the initial specification of Flk-1+/SCL+ hematovascular mesoderm. Collectively, these experiments demonstrate that Eomes sits at the top of the transcriptional hierarchy, functioning upstream of Runx1 expression and SCL functional activity, and promotes hemogenic competence of the entire YS mesodermal lineage. Overall design: EoV5 ChIP-Seq occupancy profiles for one wild type and two Eo-V5 clones (A&B) from day 4 EBs, generated by deep sequencing using Illumina HiSeq4000.

胚外中胚层（Extra-embryonic mesoderm, ExM）是最早穿过原条的细胞类群，可分化为发育中卵黄囊（yolk-sac, YS）内的内皮细胞与造血祖细胞。目前学界对ExM的特定亚群如何定向分化为造血谱系的机制仍不明确。本研究发现，T-box转录因子Eomesodermin（Eomes）在几乎可产生全部YS造血与内皮细胞的ExM祖细胞中呈瞬时表达。借助胚胎干细胞（embryonic stem cell, ESC）分化模型，我们证实Eomes的活性对于原始红细胞的生成，以及产生确定性造血祖细胞的Runt相关转录因子1（Runx1）阳性造血内皮（Runx1+ HE）的正常发育不可或缺。RNA测序（RNA-Seq）与转座酶可及性测序（ATAC-Seq）实验结果显示，Eomes可调控大量造血增强子的染色质可及性；这类增强子通常由干细胞白血病因子（Stem Cell Leukemia, SCL）介导，用于在胎肝激酶1（Flk-1）高表达/血小板衍生生长因子受体α（PDGFRα）阴性的造血血管中胚层中特化原始红细胞与造血内皮。染色质免疫沉淀测序（ChIP-Seq）实验表明，在激活素（Activin）/Nodal与Tead-Yap信号通路的调控背景下，Eomes可协同调控具有造血潜能的中胚层的发育。最后，单细胞RNA测序（scRNA-seq）结果显示，当Eomes功能缺失时，在Flk-1+/SCL+造血血管中胚层完成初始特化后，细胞会发生分化转向，偏向内皮细胞谱系而非造血细胞谱系。综上，本研究通过一系列实验证实，Eomes位于转录调控层级的顶端，作用于Runx1表达与SCL功能活性的上游，并可促进整个卵黄囊中胚层谱系的造血潜能。整体实验设计：针对第4天胚状体（embryoid bodies, EBs）的1株野生型样本与2株Eo-V5克隆（A、B），开展EoV5染色质免疫沉淀测序（ChIP-Seq）结合谱分析，通过Illumina HiSeq4000平台进行深度测序获取数据。