Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse ES cells bearing a targeted, doxycycline inducible Mesp1 transgene were differentiated in the absence or presence of DKK1

ABSTRACT: During gastrulation, cells of the prospective mesoderm ingress through the primitive streak and acquire fates based on complex spatial and temporal cues. Progenitors of the cardiogenic mesoderm are first found at E6.5 in the posterior lateral epiblast and subsequently migrate laterally and anteriorly to form the cardiac crescent at E7.5, when regionalized cell fates are first delineated . Lineage tracing and heterotopic transplantation studies suggest that precursors in the earliest heart field possess potential to generate myocardium, endocardium, and pericardium. The mechanisms by which inductive signals in the primitive streak effect the development of this pancardiac progenitor field, however, remain poorly understood; In mice, the earliest restricted progenitors of the cardiovascular system are marked by expression of the basic helix-loop-helix transcription factor, Mesp1. We therefore use microarray analysis to determine the early and intermediate effects of transiently forced Mesp1 expression during ES cell differentiation. Experiment Overall Design: ES cells bearing a targeted, doxycycline inducible Mesp1 transgene were differentiated in the absence or presence of DKK1 from day 0-4, either with or without transient doxycycline treatment from day 2-4.

ORGANISM(S): Mus musculus

SUBMITTER: Coleman Lindsley

PROVIDER: E-GEOD-5976 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human embryonic and induced pluripotent stem cells (hESCs/iPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/iPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes should be required. Though marker gene transduction or fluorescent-based purification methods were reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purposes but have not been established yet. In this study, we used microarrays to detail the global gene program during cardiac differentiation and to identify cardiac-specific cell surface markers. hiPSCs (201B6) were differentiated toward cardiomyocytes using a modified-directed differentiation protocol (high density culture in RPMI+B27-insulin, sequential administration of Activin A 100ng/mL 1 day, BMP4 10ng/mL+bFGF 10ng/mL 4 days, and Dkk1 100ng/mL 2 days). Beating clusters were first observed at day 8-9 and spread by day 11 after Activin A administration. Cardiac troponin-T (cTnT)-positive cells appeared at day 7-8 after induction and were observed in 30-70% of cells at day 11. qPCR and genome-wide analysis reflected differentiation processes from the undifferentiated state to cardiomyocytes. Rapid downregulation of pluripotent stem cell markers such as NANOG and POU5F1 was observed within 2 days of differentiation. Early and cardiac mesodermal genes (T, MESP1, KDR, ISL1) were expressed during day 2-5, and cardiac genes (NKX2-5, MYH6, MYH7, MYL2, and MYL7) were expressed after day 7. We identified VCAM1 as a cardiac-specific cell surface marker by microarray and flow cytometry. Human induced pluripotent stem cells (iPSCs; 201B6) were differentiated toward cardiomyocytes (RPMI+B27 medium supplemented d0-1 Activin A, d1-5 BMP4+bFGF, d5-7 Dkk1). RNA was extracted from cells at day 0, day 2, day 5, day 7, day 9, and day 11. Cardiomyocytes appeared after day 7 and reached about 50% of total cells at day 11.

Project description:CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger DNA binding protein with tens of thousands of binding sites genome-wide. CTCF acts as a multifunctional regulator of transcription, having been previously associated with activator, repressor, and insulator activity. These diverse regulatory functions are crucial for preimplantation development and are implicated in the regulation of numerous lineage-specific genes. Despite playing a critical role in developmental gene regulation, the mechanisms that underlie developmental changes in CTCF recruitment and function are poorly understood. Our previous work suggested that differences in CTCFM-bM-^@M-^Ys binding site sequence may affect the regulation of CTCF recruitment, as well as CTCFM-bM-^@M-^Ys regulatory function. To investigate these two possibilities directly during a developmental process, changes in genome-wide CTCF binding and gene expression were characterized during in vitro differentiation of mouse embryonic stem cells. CTCF binding sites were initially separated into three classes (named LowOc, MedOc, and HighOc) based on similarity to the consensus motif. The LowOc class, with lower-similarity to the consensus motif, is more likely to show changes in binding during differentiation. These more dynamically bound sites are enriched for motifs that confer a lower in vitro affinity for CTCF, suggesting a mechanism where sites with low-binding affinity are more amenable to developmental control. Additionally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. In sum, these results suggest that the regulatory control of CTCFM-bM-^@M-^Ys binding and function is dependent in part upon specific motifs within its DNA binding site. Mouse E14 ES cells were differentiated in vitro for 4.5 days using retinoic acid. RNA-Seq was performed from cells collected before and after differentiation.

Project description:CTCF (CCCTC-binding factor) is a highly conserved 11-zinc finger DNA binding protein with tens of thousands of binding sites genome-wide. CTCF acts as a multifunctional regulator of transcription, having been previously associated with activator, repressor, and insulator activity. These diverse regulatory functions are crucial for preimplantation development and are implicated in the regulation of numerous lineage-specific genes. Despite playing a critical role in developmental gene regulation, the mechanisms that underlie developmental changes in CTCF recruitment and function are poorly understood. Our previous work suggested that differences in CTCFM-bM-^@M-^Ys binding site sequence may affect the regulation of CTCF recruitment, as well as CTCFM-bM-^@M-^Ys regulatory function. To investigate these two possibilities directly during a developmental process, changes in genome-wide CTCF binding and gene expression were characterized during in vitro differentiation of mouse embryonic stem cells. CTCF binding sites were initially separated into three classes (named LowOc, MedOc, and HighOc) based on similarity to the consensus motif. The LowOc class, with lower-similarity to the consensus motif, is more likely to show changes in binding during differentiation. These more dynamically bound sites are enriched for motifs that confer a lower in vitro affinity for CTCF, suggesting a mechanism where sites with low-binding affinity are more amenable to developmental control. Additionally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. In sum, these results suggest that the regulatory control of CTCFM-bM-^@M-^Ys binding and function is dependent in part upon specific motifs within its DNA binding site. Mouse E14 ES cells were differentiated in vitro for 4.5 days using retinoic acid. ChIP-seq for CTCF and an IgG control was performed from cells collected before and after differentiation. For undifferentiated cells, data were generated in two biological replicates.

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Transcription profiling of mouse ES cells bearing a targeted, doxycycline inducible Mesp1 transgene were differentiated in the absence or presence of DKK1

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