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1.
Nucleic Acids Res ; 45(8): 4344-4358, 2017 05 05.
Article in English | MEDLINE | ID: mdl-28334937

ABSTRACT

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.


Subject(s)
Endothelial Cells/metabolism , Epigenesis, Genetic , GATA2 Transcription Factor/genetics , Histones/genetics , Mouse Embryonic Stem Cells/metabolism , Proto-Oncogene Protein c-ets-1/genetics , SOXF Transcription Factors/genetics , Animals , Cell Differentiation , Cell Lineage/genetics , Endothelial Cells/cytology , GATA2 Transcription Factor/antagonists & inhibitors , GATA2 Transcription Factor/metabolism , Histones/metabolism , Mice , Mouse Embryonic Stem Cells/cytology , Oligonucleotide Array Sequence Analysis , Primary Cell Culture , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Protein c-ets-1/antagonists & inhibitors , Proto-Oncogene Protein c-ets-1/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SOXF Transcription Factors/antagonists & inhibitors , SOXF Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Nat Commun ; 5: 4552, 2014 Jul 29.
Article in English | MEDLINE | ID: mdl-25072663

ABSTRACT

The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.


Subject(s)
Angiopoietin-1/metabolism , Coronary Vessels/embryology , Embryonic Stem Cells/physiology , Heart/embryology , Myocardium/metabolism , Angiopoietin-1/genetics , Animals , Cell Differentiation/physiology , Chimera , DNA Primers/genetics , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunohistochemistry , In Situ Hybridization , Mice , Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction
3.
Biochem Biophys Res Commun ; 444(2): 158-63, 2014 Feb 07.
Article in English | MEDLINE | ID: mdl-24462858

ABSTRACT

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments.


Subject(s)
CRISPR-Associated Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Knockout Techniques/methods , Pluripotent Stem Cells/metabolism , Promoter Regions, Genetic/genetics , Animals , CRISPR-Associated Proteins/metabolism , Cell Differentiation/genetics , Cell Line , Cytomegalovirus/genetics , Doxycycline/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression/drug effects , Immunoblotting , Mice , Models, Genetic , Mutagenesis, Insertional , Pluripotent Stem Cells/cytology , Reverse Transcriptase Polymerase Chain Reaction , Tetracycline/pharmacology , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
4.
Stem Cells ; 30(4): 687-96, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22267325

ABSTRACT

Ets family protein Etv2 (also called ER71 or Etsrp) is a key factor for initiation of vascular and blood development from mesodermal cells. However, regulatory mechanisms and inducing signals for Etv2 expression have been largely unknown. Previously, we revealed that cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling enhanced differentiation of vascular progenitors into endothelial cells (ECs) and hematopoietic cells (HPCs) using an embryonic stem cell (ESC) differentiation system. Here, we show that PKA activation in an earlier differentiation stage can trigger EC/HPC differentiation through Etv2 induction. We found Etv2 was markedly upregulated by PKA activation preceding EC and HPC differentiation. We identified two cAMP response element (CRE) sequences in the Etv2 promoter and 5'-untranslated region and confirmed that CRE-binding protein (CREB) directly binds to the CRE sites and activates Etv2 transcription. Expression of a dominant negative form of CREB completely inhibited PKA-elicited Etv2 expression and induction of EC/HPCs from ESCs. Furthermore, blockade of PKA significantly inhibited Etv2 expression in ex vivo whole-embryo culture using Etv2-Venus knockin mice. These data indicated that PKA/CREB pathway is a critical regulator for the initiation of EC/HPC differentiation via Etv2 transcription. This early-stage molecular linkage between a triggering signal and transcriptional cascades for differentiation would provide novel insights in vascular and blood development and cell fate determination.


Subject(s)
Cell Differentiation , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Endothelial Cells/cytology , Hematopoietic Stem Cells/cytology , Proto-Oncogene Protein c-ets-1/metabolism , Signal Transduction , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cell Line , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/enzymology , Endothelial Cells/metabolism , Enzyme Activation , Hematopoietic Stem Cells/metabolism , Mice , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Proto-Oncogene Protein c-ets-1/genetics , RNA, Small Interfering/metabolism , Tissue Culture Techniques , Transcription, Genetic
5.
J Cell Biol ; 189(2): 325-38, 2010 Apr 19.
Article in English | MEDLINE | ID: mdl-20404113

ABSTRACT

Molecular mechanisms controlling arterial-venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1(+) vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and beta-catenin signaling. Notch and GSK3beta-mediated beta-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and beta-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and beta-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.


Subject(s)
Arteries/embryology , Endothelial Cells/physiology , Receptors, Notch/metabolism , Signal Transduction/physiology , Stem Cells/physiology , beta Catenin/metabolism , Animals , Arteries/cytology , Arteries/metabolism , Biomarkers/metabolism , Cell Differentiation/physiology , Cyclic AMP/metabolism , Endothelial Cells/cytology , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mice , Neovascularization, Physiologic/physiology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Receptors, Notch/genetics , Stem Cells/cytology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Veins/cytology , Veins/embryology , Veins/metabolism , beta Catenin/genetics
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