A Gata4 nuclear GFP transcriptional reporter to study endoderm and cardiac development in the mouse.
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ABSTRACT:
SUBMITTER: Simon CS
PROVIDER: S-EPMC6310872 | biostudies-other | 2018 Dec
REPOSITORIES: biostudies-other
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Project description:The transcription factor Gata4 is essential for normal heart morphogenesis and regulates the survival, growth, and proliferation of cardiomyocytes. We tested if Gata4 can specify cardiomyocyte fate from an uncommitted stem or progenitor cell population, by developing a system for conditional expression of Gata4 in embryonic stem cells. We find that in embryoid body cultures containing even a low ratio of these cells, expression of Gata4 is sufficient to enhance significantly the generation of cardiomyocytes, via a non-cell-autonomous mechanism. The Gata4-expressing cells do not generate cardiac or other mesoderm derivatives. Rather, Gata4 expression directs the development of two types of Sox17+ endoderm. This includes an epCam+Dpp4+ subtype of visceral endoderm. In addition, Gata4 generates similar amounts of epCam+Dpp4- definitive endoderm enriched for Cxcr4, FoxA2, FoxA3, Dlx5 and other characteristic transcripts. Both types of endoderm express cardiac-inducing factors, including WNT antagonists Dkk1 and Sfrp5, although the visceral endoderm subtype has much higher cardiac-inducing activity correlating with relatively enhanced levels of transcripts encoding BMPs. The Gata4-expressing cells eventually express differentiation markers showing commitment to liver development, even under conditions that normally support mesoderm development. The results suggest that Gata4 is capable of specifying endoderm fates that facilitate, with temporal and spatial specificity, the generation of cardiomyocyte progenitors from associated mesoderm.
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Project description:BackgroundEpimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized.ResultsTo examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine (S2) residue results in negligible effects, the lack of serine and lysine (S2K3) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9-18% between corresponding polyA- and polyT-based constructs.ConclusionsHere we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.
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Project description:The embryonic endoderm is a multipotent progenitor cell population that gives rise to the epithelia of the digestive and respiratory tracts, the liver and the pancreas. Among the transcription factors that have been shown to be important for endoderm development and gut morphogenesis is GATA4. Despite the important role of GATA4 in endoderm development, its transcriptional regulation is not well understood. In this study, we identified an intronic enhancer from the mouse Gata4 gene that directs expression to the definitive endoderm in the early embryo. The activity of this enhancer is initially broad in all endodermal progenitors, as demonstrated by fate mapping analysis using the Cre/loxP system, but becomes restricted to the dorsal foregut and midgut, and associated organs such as dorsal pancreas and stomach. The function of the intronic Gata4 enhancer is dependent upon a conserved Forkhead transcription factor-binding site, which is bound by recombinant FoxA2 in vitro. These studies identify Gata4 as a direct transcriptional target of FoxA2 in the hierarchy of the transcriptional regulatory network that controls the development of the definitive endoderm.
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Project description:GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4(fl/fl); Gata6(fl/fl); Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube.
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