Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative-PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction with active repression of specific programs such as epithelial-mesenchymal transition, Hox gene activation, cell-cycle progression and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors, whereas it is dispensable for the activation of approximately half of the genes up-regulated in PGCs.
Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice.
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An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
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View SamplesThe inner cell mass (ICM) of the early blastocyst at E3.5, a source of ES cell derivation, is a morphologically homogeneous population of undifferentiated pluripotent cells that give rise to all embryonic lineages. The immediate application of the newly developed V1V3 method to single cells in this stage of mouse embryos revealed the presence of two populations of cells, one with primitive endoderm expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated primitive endoderm and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell, and developmental biology, where small numbers of distinctive or diseased cells play critical roles.
An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
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View SamplesA systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy.
An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.
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View SamplesThe contribution of chronic antigen stimulation to the occurrence of lymphoproliferative disorder (LPD) with the gamma-delta T-cell lineage is unclear, despite the fact that Epstein-Barr virus (EBV) positive T-cell LPD is derived from antigen-stimulated cytotoxic T-cells. Given the possible association of antigen stimulation with the development of cytotoxic T-cell LPD, we compared gene expression patterns in Epstein-Barr virus (EBV)-positive gamma-delta T-cell lines derived from patients with nasal T-cell lymphoma and chronic active EBV infection and those in gamma-delta T-cells from healthy volunteers. Three EBV-positive gamma-delta T-cells lines, SNT cells (SNT-8, SNT-13 and SNT-15), were used in this study. SNT-8 was established from patients with nasal T-cell lymphoma and SNT-13, -15 were established from patients with chronic active EBV infection (Zhang Y, et al., Br J Cancer 94:599-608, 2006). All the SNT cells exhibits common rearrangement of Vgamma9-JgammaP and Jdelta3 genes. The gamma-delta T-cells obtained from healthy volunteers were expanded ex vivo by 1 microM of zoledronate (ZOL) plus IL-2 for 14 days incubation.
Aberrant expression of NK cell receptors in Epstein-Barr virus-positive gammadelta T-cell lymphoproliferative disorders.
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View SamplesIron plays a central role in the regulation of many cellular functions. Dysregulation of its metabolism leads an iron overload situation and iron depletion leads to an inhibition of cell proliferation. Recent reports demonstrated that ICL670 (Novartis) acts as a potent NF-kappa-B inhibitor and improves hematological data in a subset of MDS patients (Cilloni et al, Haematologica, s1: 238, 2007). However, the precise mechanism of anti-cancer effect of ICL670 is still uncertain.
The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.
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View SamplesHeat shock protein 90 (Hsp90) is essential for the stability and the function of many client proteins, such as ERB2, C-RAF, CDK4, HIF-1 aplha and AKT. Recent reports demonstrated that inhibition of Hsp90 modulates multiple functions required for survival of human cancer, such as myeloma (Mitsiades et al, Blood:107, 1092, 2006), The aim of this study is evaluate the effect of Hsp90 inhibition, and to identify molecular pathways responsible for anti-proliferative effect on ATL cells. For Hsp90 inhibition, Geldanamycin derivates, 17AAG (17-allylamino -17-demethoxygeldanamycin) and 17DMAG (17-(dimethylaminoethylamino) 17-demethoxygeldanamycin) were used in this study. Interleukin 2-independent ATL cell lines (MT-2 and MT-4) and an interleukin 2-dependent ATL cell line (TaY-E10) were incubated, with or without Hsp90 inhibitors.
Anti-proliferative activity of heat shock protein (Hsp) 90 inhibitors via beta-catenin/TCF7L2 pathway in adult T cell leukemia cells.
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View SamplesAdult T-cell leukemia (ATL) is a fatal neoplasia derived from HTLV-1 infected T lymphocytes exhibiting constitutive activation of NF-kB. To elucidate the complex molecular mechanism of anti-tumor effect of the proteasome inhibitor, bortezomib in ATL cells, we attempted to perform gene expression profiling.
Induction of heme oxygenase-1 by cobalt protoporphyrin enhances the antitumour effect of bortezomib in adult T-cell leukaemia cells.
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View SamplesPlacental development is a key event in mammalian reproduction and embryogenesis. However, the molecular basis underlying extraembryonic lineage specification and subsequent placental development is not fully understood. Through a genetic screen, we identified Zfp281 as a key factor for extraembryonic development. Disruption of Zfp281 in mice caused severe defects in extraembryonic as well as embryonic tissues. Importantly, Zfp281 was preferentially expressed in the trophoblast stem cell population in an FGF-dependent manner and ensured the expression of genes necessary for placental development. Through the analysis of transcriptome and epigenome, we identified Zfp281 as an important factor to shape the transcriptome of mammalian trophoblast stem cells. Overall design: To study the role of Zfp281 in transcriptional regulation, we performed RNA-seq using mouse and human TS cells. Furthermore, we performed H3K4me3 ChIP-seq and ATAC-seq to reveal the roles of Zfp281 in chromatin regulation.
Zfp281 Shapes the Transcriptome of Trophoblast Stem Cells and Is Essential for Placental Development.
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View SamplesStem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs following the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or over-expression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach we have, for the first time, formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.
Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment.
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