Project description:Epicardial cells are progenitors giving rise to the majority of cardiac fibroblasts, coronary smooth muscle cells, and pericytes during cardiac development, and critically modulating heart morphogenesis and coronary development. An integral phase of epicardial cell fate transition is epithelial-to-mesenchymal transition (EMT), which confers motility and facilitates cell fate transition. We identify a pathway involving protein arginine methyltransferase 1 (PRMT1) and its downstream p53 signaling that drives epicardial EMT and invasion. We show that PRMT1 determines the half-life of p53 through regulating alternative splicing of Mdm4, which is a key controller of p53 degradation. Loss of PRMT1 promotes the expression of Mdm4 short form, which inhibits p53 degradation. Accumulation of p53 subsequently enhances Slug degradation and blocks epicardial EMT. We further demonstrated that the PRMT1-Mdm4-p53 pathway drives epicardial cell fate transition into cardiac fibroblasts, coronary smooth muscle cells and pericytes in vivo, and modulates ventricular morphogenesis and coronary vessel formation. Together, our results establish critical functions of the PRMT1-Mdm4-p53 pathway in epicardial EMT, invasion and cell fate transition.
Project description:Epicardial cells undergo an epithelial-to-mesenchymal transtion (EMT) to generate coronary vascular smooth muscle cells (VSMC) and cardiac fibroblasts. Little is known about the mechanisms regulating EMT or the in vivo signals directing epicardial-derived cell (EPDC) fate. Here, we show that loss of PDGF signaling leads to a disruption in Sox9 expression, and when Sox9 expression was restored in mutant hearts, the EMT defect was rescued. Interestingly, mutants lacking only one of the PDGF genes exhibited a lineage specific requirement for the individual receptors. Loss of PDGFRα resulted in a deficit in cardiac fibroblast formation, while cVSMC development was unperturbed. Conversely, PDGFRβ was required for cVSMC development but not cardiac fibroblast development. Combined, our data demonstrate a novel role for PDGF receptors in epicardial EMT and EPDC development. GSM671723-GSM671724: Total RNA was isolated from E12.5 control and PDGF receptor epicardial knockout hearts using the Trizol reagent. RNA was processed as per manufacturer's instructions (Illumina Gene expression array, Illumina, inc. San Diego, CA, USA) GSM671877-GSM671882: Total RNA was isolated from E12.5 control and PDGF receptor epicardial knockout primary epicardial cultures using the Trizol reagent. RNA was processed as per manufacturer's instructions (Illumina Gene expression array, Illumina, inc. San Diego, CA, USA)
Project description:Mice lacking the zinc finger transcription factor Specificity protein 3 (Sp3) die prenatally in the C57Bl/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at E10.5, and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analysed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of Cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. ChIP analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development, and suggest that it has a crucial role in myocardial differentiation. ( Keywords: Transcription factors, Sp3, knockout mice, cardiac malformations, E12.5
Project description:Identification of epicardium-enriched genes in the embryonic heart. The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in re-activation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration. Total RNA obtained from lacZ-positive epicardial cells isolated from the E11.5 Tcf21lacZ hearts compared to total dissociated heart cells
Project description:We found that cardiomyocyte-specific PRMT1-deficient (PRMT1-cKO) mice showed dilated cardiomyopathy and aberrant cardiac alternative splicing. To identify novel cardiac splicing events, we performed a comprehensive analysis of gene expression changes in hearts of wildtype (WT) and PRMT1-cKO mice using RNA sequencing (RNA-Seq). To investigate differentially expressed genes (false discovery rate (FDR) p<0.05, fold change >2) between control and PRMT1-cKO mice, we performed pairwise comparisons of RNA-Seq data using the CLC Genomics Workbench software.
Project description:Purpose: to identify genes directly bound by HIF1a in cardiomycoytes Methods: crosslinked chromatin isolated from E12.5 mouse hearts was precipitated using an anti-HIF1a antibody and processed for sequencing. A list of genes containing a HIF1a peak (Minimum score 3.5) was compared with RNA-seq results to identify genes that are bound by HIF1a and expressed in E12.5 hearts Results: this analysis identified 1016 genes expressed and bound by HIF1a in embryonic hearts
Project description:In order to identify the targets of GATA4-FOG2 action in mammalian heart development we performed Affymetrix microarray comparisons of gene expression in normal and mutant at embryonic (E) day E12.5 hearts. We compared RNA samples from both Fog2-null and Gata4ki/ki mutant E12.5 hearts to the wild-type control E12.5 hearts. We reasoned that as the phenotypes of the Fog2 knockout and Gata4ki/ki mutation (a V217G mutation that specifically cripples the interaction between GATA4 and FOG proteins) are similar, we should expect to identify a similar set of differentially expressed genes in both experiments. As an additional control, we expected to find the Fog2 gene expression absent in the mutant (null) Fog2 cardiac sample, but not Gata4ki/ki sample.
Project description:In order to identify the targets of GATA4-FOG2 action in mammalian heart development we performed Affymetrix microarray comparisons of gene expression in normal and mutant at embryonic (E) day E12.5 hearts. We compared RNA samples from both Fog2-null and Gata4ki/ki mutant E12.5 hearts to the wild-type control E12.5 hearts. We reasoned that as the phenotypes of the Fog2 knockout and Gata4ki/ki mutation (a V217G mutation that specifically cripples the interaction between GATA4 and FOG proteins) are similar, we should expect to identify a similar set of differentially expressed genes in both experiments. As an additional control, we expected to find the Fog2 gene expression absent in the mutant (null) Fog2 cardiac sample, but not Gata4ki/ki sample. We have analyzed 6 RNA samples total (3 from control hearts, 2 from FOG2 null hearts and 1 from GATA4ki hearts)
Project description:To investigate roles for Tbx20 in endocardium, we ablated Tbx20 utilizing Tie2Cre. Tie2Cre;Tbx20 mutants died at E14, exhibiting defects in multiple aspects of cardiac septation. Although endocardial cells lacking Tbx20 were able to undergo endothelial-to-mesenchymal transition, cushion mesenchymal cells lacking Tbx20 did not disperse normally. Non-cell autonomous roles of endocardial Tbx20 were also revealed, as evidenced by decreased myocardialization of outflow tract and failure of dorsal mesenchymal protrusion formation in mutants. To examine how ablation of Tbx20 in endocardial lineages affected gene expression, we performed global gene expression analysis on purified endocardial lineages. E12.5 hearts were dissociated, and Tie2Cre;RosatdTom lineage traced cells of controls and mutants were isolated by fluorescence activated cell sorting (FACS), after exclusion of blood cells (Ter119+, CD41+ and/or CD45+). Mutant endocardial lineages exhibited decreased expression of genes associated with extracellular matrix and cell migration. E12.5 hearts were dissociated, and Tie2Cre;RosatdTom lineage traced cells of controls and mutants were isolated by fluorescence activated cell sorting (FACS), after exclusion of blood cells (Ter119+, CD41+ and/or CD45+). FACS sorted Tie2Cre lineage from E12.5 hearts: Tie2Cre;Tbx20 +/loxP Control hearts versus Tie2Cre;Tbx20 loxP/- mutant hearts