15 research outputs found
Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells
No full textRequirement of Oct3/4 function for germ cell specification
No full textAbstractIn mammalian embryos, PGCs (primordial germ cells) are specified from a pluripotent epiblast cell population after implantation. In this study, we demonstrated an essential role for the germline-specific transcription factor Oct3/4 in PGC specification. We generated chimeric embryos with ZHBTc4 ES cells lacking both alleles of the Oct3/4 gene (pou5f1). Pluripotency was maintained by an Oct3/4 transgene, and its expression was suppressed by doxycycline (Dox). Transcription of the Oct3/4 transgene in the ES-derived cells unexpectedly suffered constitutive suppression in chimeric embryos without Dox, and the ES-derived cells contributed to PGC precursor-like cells, but failed to form PGCs. We then attempted to rescue Oct3/4 expression in the ES-derived cells in the chimeric embryos by introducing an additional Oct3/4 transgene. The ES cell-derived cells indeed recovered Oct3/4 transcription in these chimeric embryos, and were successfully specified to PGCs. We further confirmed the requirement of Oct3/4 by using another derivative of ZHBTc4 ES cells in which a Dex (dexamethasone)-dependent Oct3/4 transgene was introduced. In the presence of Dox, Oct3/4 protein was absent in the nuclei of the ES-derived cells, which failed to form PGCs. In contrast, the ES-derived cells could be specified to PGCs after activation of Oct3/4 function in the presence of Dex
Heterogeneity of mouse primordial germ cells reflecting the distinct status of their differentiation, proliferation and apoptosis can be classified by the expression of cell surface proteins integrin α6 and c-Kit
No full textImplication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells.
No full textPrimordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs
DNA demethylation of the regulatory region of <i>mil-1</i> resulted in upregulation of its expression in ES cells.
No full text<p>(A) Bisulfite sequencing analysis of the regulatory region of <i>mil-1</i> and (B) quantitative RT-PCR analysis of <i>mil-1</i> expression were performed on embryonic stem (ES) cells with or without siRNA-mediates <i>Dnmt1</i> knockdown (<i>Dnmt1</i> KD/Con KD). (A) The regulatory region became more hypomethylated following <i>Dnmt1</i> knockdown. (B) Histogram represents the relative expression level of <i>mil-1</i> in the <i>Dnmt1</i>-knockdown ES cells. The expression level in the control ES cells (Con KD) was set as 1.0. <i>Gapdh</i> PCR signal was used an internal control to measure relative expression. The data were obtained from four independent experiments. *p<0.05. Error bars represent SEM. (C) Luciferase activity of the reporter vectors with methylated or unmethylated regulatory region of <i>mil-1</i> in ES cells. Luciferase activity was normalized against the activity of a cotransfected <i>Renilla</i> construct. The liciferase activity of the methylated construct (Methylated 3.0k-pCpGL) was set as 1.0. The data were obtained from six independent experiments. *p<0.05. Error bars represent SEM.</p
Bivalent histone modification on the somatic genes in PGCs.
No full text<p>(A, B) ChIP analysis with the H3K4me3 or H3K27me3 antibodies for the promoter regions of somatic genes (<i>Hoxa1</i>, <i>Hoxb1</i>, and <i>Gfap</i>) and of the PGC-specific genes (<i>mil-1</i>, <i>Blimp1</i>, and <i>Stella</i>) was performed on EpiSCs (A), and male and female PGCs at E13.5 (B), showing the bivalent histone modification. Histogram represents ratios of the immuno-precipitated chromatin to the input chromatin, which was quantified by quantitative PCR analysis. Also shown are results using beads only as a no antibody control (NAC). Shown is a representative data from two independent experiments.</p
The regulatory region of <i>mil-1</i> becomes hypomethylated during PGC development.
No full text<p>Bisulfite sequencing analysis of the regulatory region of <i>mil-1</i> was performed using epiblasts, PGCs/somatic cells purified as GFP positive/negative cells from embryos at each embryonic day (E). The rectangle containing <i>mil-1</i> in the top line represents an exon, and the numbers with ‘kb (kilobase)’ under the line indicate distance from the transcription start site (TSS). The box outlined in green represents the regulatory element required for PGC-specific expression and the <i>Ifitm</i> genes consensus element (ICE) is shown in more detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046036#pone-0046036-g004" target="_blank">Figure 4A</a>. Each circle corresponds to a CpG site in the regulatory region, and the degree of gray in each circle corresponds to the level of DNA methylation.</p
Knockdown of <i>Dnmt1</i> causes hypomethylation of <i>Blimp1</i> and <i>Stella</i> flanking regions and upregulation of <i>Blimp1</i> and <i>Stella</i> expression in ES cells.
No full text<p>(A) Bisulfite sequencing analysis of the flanking regions of <i>Blimp1</i> and <i>Stella</i> and (B) quantitative RT-PCR analysis of <i>Blimp1</i> and <i>Stella</i> expression were performed using ES cells with or without <i>Dnmt1</i> knockdown treatment (<i>Dnmt1</i> KD/Con KD).</p
The expression of <i>mil-1</i>, <i>Blimp1</i>, and <i>Stella</i> become upregulated during PGC development.
No full text<p>(A, B, C) Quantitative RT-PCR analysis of the expression of (A) <i>mil-1</i>, (B) <i>Blimp1</i>, and (C) <i>Stella</i> was performed using epiblasts (E6.0) and PGCs (E7.5 and E9.0). Histograms represent relative expression levels of these three genes at each developmental stage. The averages of expression levels in the epiblasts (E6.0) were set as 1.0. <i>Gapdh</i> PCR signal was used as an internal control to measure relative. The data were obtained from three individual embryos. *p<0.05. Error bars represent SEM.</p
Knockdown of <i>Dnmt1</i> causes upregulation of <i>mil-1</i> and <i>Stella</i> expression in PGCLCs.
No full text<p>Quantitative RT-PCR analysis of <i>mil-1</i> and <i>Stella</i> expression in PGCLCs (PGC-like cells) with or without <i>Dnmt1</i> knockdown (<i>Dnmt1</i> KD/Con KD). The expression level in EpiLCs was set as 1.0. Shown is a representative data from two independent experiments.</p
