Comparison of Hob6-RFP positive and negative Flk-1+ cells (RFP reporter)
ABSTRACT: ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by RFP Hoxb6 reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-RFP positive and negative Flk-1+ cells.
Project description:ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by Venus reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-Venus positive and negative Flk-1+ cells.
Project description:Screening for genes up in Etv2+ cells within Flk-1+ ES derived mesoderm Microarray analysis performed to screen for the candidate genes regulated by Etv2. Differentiated Flk-1+ mesoderm can be devided into Etv2+ or-. Etv2+ cells are assumed to be committed to hemato/endothelial cells. Comparison of two populations can reveal genes relevant in this commitment. Extract RNA from sorted Flk-1+/Etv2- vs Flk-1+/Etv2+ populations.Etv2-Venus KI ES cells were differentiated on OP9 for 4-5 days and Flk-1+ population was separated into Etv2-Venus+ or- cells. Total RNA was purified from each population for analysis.
Project description:Screening for genes regulated by Etv2 within Flk-1+/PDGFRa+ ES derived mesoderm.Microarray analysis performed to screen for the candidate genes regulated by Etv2. TT2 ES cells differentiated on OP9 feeder cells were sorted using Flk-1 and PDGFRa antibodies.Gene expressions from these two populations were compared. Extract RNA from sorted Flk-1+/PDGFRa+ populations from Etv2Het vs KO cells. To obtain primitive mesoderm cells TT2 ES cells of corresponding genotypes were differentiated on OP9 cells for 4 days. Flk-1+/PDGFRa+ populations were sorted from Etv2 Het vs. KO cells for RNA extraction.
Project description:Ets transcription factor ER71 is critical for Flk-1 mesoderm specification to different cell lineages. In this dataset, we determine to investigate the mechanisms by which ER71 regulates hematopoietic and endothelial cell versus cardiac cell lineage development. Expression of all four Flk-1+ mesoderm populations (i.e. Er71 overexpressed versus control, Er71 deficient versus control Flk-1+ mesoderm) was compared. Specifically, we sorted Flk-1+ mesoderm from day 3 differentiated embryonic stem(ES) cells, including induced Er71 and control ES cells, as well as Er71+/+ as well as Er71-/- ES cells .
Project description:Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng+Flk1+ mesodermal precursor population at E7.5, a cell fraction also endowed with endothelial potential. In Eng knockout embryos, hematopoietic colony activity and numbers of CD71+Ter119+ erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key BMP target genes including the hematopoietic regulators Scl, Gata1, Gata2 and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng+Flk-1+ progenitors co-expressed TGFβ and BMP receptors and target genes. Furthermore, Eng+Flk-1+ cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of endoglin-expressing cells was dependent on TGFβ superfamily ligands: BMP4, BMP2, or TGF-β1. These data demonstrate that the E+F+ fraction at E7.5 represents mesodermal cells competent to respond to TGFb1, BMP4, or BMP2, shaping their hematopoietic development, and that endoglin is a critical regulator in this process by modulating TGF/BMP signaling. E7.5 pooled embryos (25 litters; 300 embryos approximately) were dissected and 3,000 cells were sorted in triplicate for Eng-Flk1-, Eng-Flk1+, Eng+Flk1+, and Eng+Flk1- fractions. Microarray results were analyzed with GeneSpring GX software.
Project description:Although differentiation of mice embryonic stem cells into vascular endothelial cells (ECs) gives a model for investigating molecular mechanisms of vascular development in vivo, temporal dynamics of gene expressions and chromatin modifications have not been studied until now. Here, we interrogated transcriptome and two histone modifications, H3K4me3 and H3K27me3, with a genome-wide scale during ECs differentiation and elucidated epigenetic switch peculiar to ECs. We find Gata2, Fli1, Sox7, and Sox18 are master regulators from genetic and epigenetic data, these genes were induced after Etv2 activation. These genes have specific histone modification pattern which is repressed by H3K27me3 modification at Flk-sorted mesoderm and changed to the bivalent (H3K4me3 and H3K27me3 both positive) state rapidly after vascular endothelial cells growth factor (VEGF) stimuli. Using a previously reported ECs differentiation model, we demonstrate that four transcription factors are critical for ECs specific gene expressions and efficient differentiation. Moreover, from knockdown experiments using si-RNA, we discovered these factors inhibited not only TGFβ signaling pathway, that is endothelial mesenchymal transition pathway, but also other near lineage commitment, including blood cells, skeletal muscle cells, vascular smooth muscle cells, and cardiomyocytes. We further identify each factor specific target genes during ECs differentiation by microarray, including both activating and repressing genes. Together, our findings from a detailed epigenetic approach provide a basic understanding temporal regulated chromatin signatures and resulting gene expression profile during ECs commitment, which is applicable to other models of differentiation and production of mature and long lasting ECs for regenerative medicine. Total 17 samples were derived from  ES cells, Flk-sorted mesoderm cells, and in the absense or presence of VEGF (6, 12, 24, and 48h) to determine VEGF activated genes during endothelial cells differentiation,  control si-RNA, si-Gata2, si-Fli1, si-Sox7, or si-Sox18 transfected cells under VEGF stimuli,  control si-RNA or si-Mix (si-Gata2, si-Fli1, si-Sox7, and si-Sox18) transfected cells under VEGF stimuli for the identification of each transcription factor dependent genes during endothelial cells differentiation.
Project description:Previous studies have demonstrated that distinct progenitor subpopulations of mesoderm display tissue specific and vascular potential: hemangioblasts, a progenitor population capable of generating cells of the hematopoietic, endothelial and vascular smooth muscle lineages, and a multipotential progenitor capable of generating progeny of the cardiac, endothelial and vascular smooth muscle lineages. Each of these populations is characterized by co-expression of brachyury (Bry) and Flk-1, although the hemangioblast population is established before the cardiovascular progenitors in ES cell differentiation cultures (e.g. d3.5 for hemagioblast, versus d4.5 for cardiovascular progenitors). To investigate the role of Notch signalling in the establishment of cardiac lineages, we used a tet-inducible ES cell line (Ainv18) engineered to express an activated form of the Notch4 receptor following doxycycline treatment. This line also expresses a GFP cDNA from the Bry locus. Following 3.0-3.5 days of serum stimulation, three distinct populations based on Flk-1 and GFP expression are observed: Bry-GFP-/Flk-1-, Bry-GFP+/Flk-1- and Bry-GFP+/Flk-1+ cells. Previous studies have shown that the Bry-GFP+/Flk-1+ population contains hemangioblasts, whereas the Bry-GFP+/Flk-1- population displays cardiac potential. Bry-GFP+/Flk-1+ cells, sorted from EB's derived from ES cell differentiation cultures exposed to serum for 3.5 days, were allowed to reaggregate for 24 in the presence or absence of doxycycline, and the total RNA harvested at 4, 12, 24, 48, and 96 hours post Dox induction for microarray analysis. The induced populations were compared to non-induced population harvested at the same time points.
Project description:We used a murine ES cell line in which HoxA3 expression is under control of a tetracycline-responsive element and differentiated these cells as embryoid bodies (EBs). Endothelial (Flk-1 VE-cadherin double positive, FV) and hematopoieitc progenitors (c-Kit CD41 double positive, K41) were isolated from differentiated EBs that had been induced for 6 hours by doxycycline (Dox) treatment. Here we show genes regulated upon HoxA3 upregulation (6 hours doxycycline treatment) in hematopoietic and endothelial progenitors.