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: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.
Project description:A dual biomarker signature extracted a stage-specific cytotype according to cell surface expression of CXCR4/FLK-1 after 5 days of spontaneous differentiation from pluripotent stem cells. Genome-wide microarray analysis revealed a high degree of similarity between CXCR4+/FLK-1+ and CXCR4-/FLK-1- subpopulations at day 5, yet the divergent gene expression profile represents more than 700 unique transcripts. Functional analysis of the 294 up-regulated and 440 down-regulated transcripts that distinguished CXCR4+/Flk-1+ from CXCR4-/Flk-1- subpopulations identified an overt ontologic prioritization of “Cardiovascular Development”-IPA 7.0, 2009.Thus, a biomarker-selected subpopulation from spontaneously differentiated pluripotent stem cells identifies a pool of genes that non-stochastically integrate into a blueprint providing instructions for cardiac lineage-specification. Keywords: Comparison of day 5 embryonic stem cell progenity: CXCR4/FLK-1 biomarker positive versus biomarker negative cells Differentiating embryonic stem cells were FACS sorted at day 5 based on a dual CXCR4/FLK-1 biomarker signature and double positive and double negative progeny were thus collected. Day 5 sorted progeny were independently collected to provide raw material for three biological replicates for each experimental condition. In this manner, three CXCR4/FLK-1 double positive biological samples, and three CXCR4/FLK-1 double negative biological samples were obtained. Total RNA was extracted from each of the samples and RNA pools were profiled on Affymetrix Mouse 430 2.0 Arrays to identify global gene expression changes between double positive and double negative progeny at day 5 of differentiation.
Project description:A dual biomarker signature extracted a stage-specific cytotype according to cell surface expression of CXCR4/FLK-1 after 5 days of spontaneous differentiation from pluripotent stem cells. Genome-wide microarray analysis revealed a high degree of similarity between CXCR4+/FLK-1+ and CXCR4-/FLK-1- subpopulations at day 5, yet the divergent gene expression profile represents more than 700 unique transcripts. Functional analysis of the 294 up-regulated and 440 down-regulated transcripts that distinguished CXCR4+/Flk-1+ from CXCR4-/Flk-1- subpopulations identified an overt ontologic prioritization of “Cardiovascular Development”-IPA 7.0, 2009.Thus, a biomarker-selected subpopulation from spontaneously differentiated pluripotent stem cells identifies a pool of genes that non-stochastically integrate into a blueprint providing instructions for cardiac lineage-specification. Keywords: Comparison of day 5 embryonic stem cell progenity: CXCR4/FLK-1 biomarker positive versus biomarker negative cells
Project description:Alternative splicing is critical for development. However, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-specific RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes likely arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Collectively, our results thus uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.
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.
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: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
Project description:Serine/threonine kinase 40 (Stk40) was previously identified as a direct target gene of pluripotency-associated transcription factor Oct4 and its overexpression could facilitate differentiation of mouse embryonic stem cells (mESCs) towards the extraembryonic endoderm. Stk40-/- mice are lethal at the perinatal stage, displaying multiple organ failures. However, the molecular mechanisms underlying the physiological functions of Stk40 remain elusive. Here, we report that Stk40 ablation compromises the mesoderm differentiation from mESCs in vitro and in embryos. Mechanistically, Stk40 interacts with both mammalian constitutive photomorphogenic protein 1 (Cop1) and c-Jun, promoting degradation of c-Jun. Consequently, Stk40 knockout leads to c-Jun protein accumulation, which, in turn, might suppress the Wnt signaling activity and impair the mesoderm differentiation process. Overall, this study reveals that Stk40, together with Cop1, represent a novel axis for modulating c-Jun protein levels within an appropriate range during mesoderm differentiation from mESCs. Our finding provides new insight into the molecular mechanism regulating c-Jun protein stability and may have potential for managing related cellular disorders.