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Single-cell RNA-seq analysis of wildtype and ETV2-overexpressing embryoid body-derived progenitor cells

ABSTRACT: Wild-type (WT) and Etv2-overexpressing (OE) mouse embryonic stem cells (mESC) were cultured and differentated to embryoid bodies (EBs) in mesodermal conditions. Induction of Etv2 in OE EBs was achieved by the addition of doxycycline. Cells were collected at different time points (i.e., D2, D3, D4 for WT and D3, D4 for OE EBs) following FACS based on the presence or absence of two surface markers: Flk1 (F+/-) and Pdgfrα (P+/-). We performed single-cell RNA sequencing (scRNA-seq) on the FACS sorted cells at different time points. The transcriptomic profiles revealed that ETV2 overexpression promotes HE lineage specification while repressing cardiac and skeletal muscle programs.

ORGANISM(S): Mus musculus

PROVIDER: GSE298879 | GEO | 2025/06/03

REPOSITORIES: GEO

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ATAC-seq analysis of wildtype and ETV2-overexpressing embryoid body-derived progenitor cells

Project description:Wild-type (WT) and Etv2-overexpressing (OE) mouse embryonic stem cells (mESC) were cultured and differentated to embryoid bodies (EBs) in mesodermal conditions. Induction of Etv2 in OE EBs was achieved by the addition of doxycycline. Cells were collected at different time points (i.e., D2, D3, D4 for WT and D3, D4 for OE EBs) following FACS sorting based on the presence or absence of two surface markers: Flk1 (F+/-) and Pdgfrα (P+/-). ATAC-seq was performed using 50,000 cells and the Tn5 transposase enzyme. Sequencing of libraries was carried out on the Illumina NextSeq platform (2×50 bp). Chromatin accessibility profiling revealed that Etv2 overexpression during EB differentiation increases accessibility of genes related to the hematoendothelial lineage, while reducing accessibility of genes associated with the cardiac lineage.

2025-06-16 | GSE299629 | GEO

Genome-wide analysis of ETS factor ER71/ETV2 chromatin occupancy

Project description:We discover that ER71/ETV2 initiates hemangiogenic program by activating blood and endothelial cell lineage specifying genes while enhancing FLK1 expression and expanding hemangioblast population. Furthermore, ER71/ETV2 establishes an ETS hierarchy by directly activating Ets genes in hematopoietic and endothelial cell lineage development. As such, ER71/ETV2-initiated blood and endothelial cell program is maintained by ER71/ETV2 downstream ETS factors through an ETS switching mechanism. ChIP-seq analysis of ER71 in differentiated embryonic stem cells

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Genome-wide analysis of gene expression of Flk1Cre;ETV2 CKO mouse yolk sacs.

Project description:To identify genes critical for vascular development, we generated mice where ETV2 is inactivated in FLK1+ cells by a loxP-Cre recombination approach. Results provide a detailed insight into the function of ETV2 in emrbyonic vasculare formation. Total RNA obtained from E9.5 yolk sacs from Flk1Cre;ETV2 CKO and control mice.

2014-11-17 | E-GEOD-51407 | biostudies-arrayexpress

Genome-wide analysis of ETS factor ER71/ETV2 chromatin occupancy

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ATAC-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ATAC-seq analysis of Etv2 induced ES/EB, MEFs,Brg1 KO EBs and Brg1 KD MEFs. Methods: EBs were collected at different time points and disaggregated in 0.25% trypsin at 37C for 3 min incubation with gentle agitation followed by inactivation with culture medium containing 10% FBS. Cells were collected by centrifugation at 500g for 5 minutes, washed once with ice-cold PBS. iHA-Etv2 MEFs were harvested from the culture dishes at different time points by treating the cells with 0.25% trypsin at 37C for 4 min incubation followed by neutralizing and collecting the cells with culture medium containing 10% FBS. ATAC reaction was performed with 50,000 cells as previously described18 using the Tn5 transposase (Illumina) and libraries were created at the University of Minnesota Genome Center. Libraries were then sequenced on a NextSeq Illumina platform (2x50 bp) aiming for 25 million reads per sample.

2022-01-12 | GSE168636 | GEO

Etv2-mediated hemangiogenic fate commitment of mesoderm

Project description:A comprehensive understanding of a lineage map and molecular mechanisms underlying lineage specification is fundamental to development. To this end, ETS transcription factor Etv2 functions as the master regulator of hematopoietic and endothelial cell formation. As such, Etv2 regulated hemangiogenesis provides an excellent model for assessing cell lineage specification. Herein, we generated several reporter embryonic stem (ES) cell lines to map developmental route pertaining to hemangiogenesis. We performed transcriptome analysis and high throughput CRISPR screening to further delineate molecular mechanisms regulating hemangiogenic lineage specification. We show a distinct lineage map of hemangiogenesis, in which hemangiogenic fate is specified not simply by the onset of Etv2 expression, but in a threshold-dependent manner. Importantly, VEGF-FLK1 signaling is necessary for efficiently achieving ETV2-threshold. Moreover, we find forkhead transcription factor Foxh1 to be required for FLK1 mesoderm formation. These studies provide a roadmap in hematopoietic and vascular cell generation and applications in regenerative medicine.

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Genome-wide analysis of gene expression of Flk1Cre;ETV2 CKO mouse yolk sacs.

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ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

Project description:ETV2/ER71, an ETS transcription factor, is critical for hematopoiesis and vascular development. However, knowledge on the molecular mechanisms behind ETV2-mediated gene transcription is limited. Here, we show that ETV2 together with KDM4A, an H3K9 demethylase, regulates hematopoietic and endothelial genes. Etv2-/- mouse embryonic stem cells (mESCs), which fail to generate hematopoietic and endothelial cells, showed enhanced levels of H3K9me3 on hematopoietic and endothelial genes. ETV2 interacts with KDM4A and the ETV2-mediated transcriptional activation of hematopoietic and endothelial genes is dependent on KDM4A histone demethylase activity. ETV2 and KDM4A co-occupy the transcription regulatory regions of genes whose expression is directly regulated by ETV2. Mice lacking Kdm4a and Etv2 in endothelial cells (Cdh5Cre;Kdm4af/f;Etv2f/f) displayed a more severe defect in perfusion recovery and neovascularization compared with Cdh5Cre;Kdm4af/f, Cdh5Cre;Etv2f/f mice and controls. Collectively, we demonstrated that ETV2 interacts with KDM4A and that this interaction is critical for FLK1+ cell generation, differentiation into the downstream lineages, and vascular regeneration.

2024-12-10 | GSE251753 | GEO

Gene expression analysis of inducible ES cells overexpressing Etv2 (induced for 12 hours at day 3 of differentiation)

Project description:During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages, however, the precise roles of Etv2 in yolk sac development remains unclear. We carried out a transcriptome analysis using an ES cell line that can inducibly express Etv2. Cells were induced for 12 hours at day 3 of differentiation. An ES cell line (dervied from mouse of background strain 129P2/Ola) with a doxycycline inducible expression casette of Etv2 (ER71 A2Lox.cre) was differentiated in hanging drops. At 3 days of differentiation, cells were treated with doxycycline for 12 hours, and subsequently Flk1(+) cells were sorted. Experiment was done in triplicate and untreated cells were used for control.

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