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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE139998 | GEO | 2020/11/01

REPOSITORIES: GEO

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Project description:Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood. To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4 and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2 additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and endothelial cells. Keywords: Comparison of induced differetiated state to original cell line. Generation of Hemangioblast and Blast Colony cell populations: Scl+/hCD4 ES cells (Chung et al., Development, 2002) were maintained on a layer of mitotically inactivated STO cells in DMEM media in the presence of LIF. Two days before differentiation, the cells were transferred to gelatinized tissue culture flasks and the media was changed to IMDM-based. ES cells were differentiated in liquid-differentiation media in the presence of FCS for 2.75 days. D2.75 EBs were recovered, dissociated in Trypsin/EDTA and stained with antibodies against Flk-1 and human CD4. Cells were then flow sorted on the basis of Flk-1 and Scl expression and the hemangioblast population was considered to be Flk-1+Scl+. D2.75 EB cells were replated as described (Chung et al., 2002) and individual Blast colonies were picked and pooled, and RNA was generated with TRIzol according to the manufacturers protocol. RNA (20 ug) was given to the Washington University Array core were it was reverse transcribed and cRNA synthesized. Replicated of the two populations (hCD4, Blast) were made. Pairwaise comparisons were performed to identify enriched genes.

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

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