Project description:DNA methylation is extensively reprogrammed during early phases of mammalian development yet individual genomic targets of this process are largely unknown. We optimized MeDIP (Methylated DNA Immunoprecipitation) for low numbers of cells and profiled DNA methylation genome-wide during early development of the mouse embryonic lineage in vivo. We mapped DNA methylation at 3 consecutive stages of early development: E3.5 blastocysts, E6.5 epiblasts and E9.5 whole embryos. MeDIP and Input samples were hybridized to Nimblegen HD2 MM8 promoter deluxe arrays covering 12 kb of all gene promoters. Experiments were performed in duplicates for E3.5 blastocysts and triplicates for E6.5 epibalsts and E9.5 embryos. As a control we also hybridized pooled unamplified MeDIPs from E9.5 to Nimblegen 385K MM8 RefSeq promoter arrays.
Project description:We quantified the targets and kinetics of DNA methylation acquisition in mouse embryos, and determined the contribution of the de novo methyltransferases DNMT3A and DNMT3B to this process. We provide single-base maps of cytosine methylation by RRBS from the blastocysts to post-implantation stages and in embryos lacking DNMT3A or DNMT3B activity, and performed RNA-Seq in embryos lacking DNMT3B activity. We sequenced RRBS libraries prepared from genomic DNA isolated from embryos at consecutive stages of development between E3.5 and E11.5,and adult differentiated cells (sperm, liver). We performed RRBS on blastocysts at E3.5/E4.5, dissected epiblasts at E5.5/E6.5/E7/5, whole embryos at E8.5/E10.5 and limbs at E11.5. RRBS experiments in Dnmt3a-/- and Dnmt3b-/- embryos were performed in biological duplicates on individual embryos. We sequenced RNA-Seq libraries prepared from total RNAs of three WT and Dnmt3b-/- littermate embryos collected at E8.5.
Project description:We demonstrated that DNMT3a was decreased at E3.5 to E6.5 stage in embryos developing from zygotes injected with sperm sRNAs from depressed mice.To uncover the consequence caused by DNMT3a transient reduction in developing embryos, we injected sperm sRNAs from normal or depressed mice into zygotes and systematically profiled the DNA methylome of embryos at E4.5 stage (sRNA-Dep-E4.5 vs sRNA-Ctl-E4.5) by single-cell level whole genome bisulphite sequencing (SC-WGBS).
Project description:In mammals, chromatin marks at imprinted genes are asymmetrically inherited from each parent to control gene expression. Many genomic imprints are determined by differentially methylated regions (DMRs), but these have not been comprehensively mapped physically or functionally in mouse preimplantation embryos. To address this, we measured genome-wide DNA methylation in individual haploid uniparental parthenogenetic haploid (ph) and androgenetic haploid (ah) E3.5 blastocysts by micro-whole-genome bisulfite sequencing (µWGBS). For comparison, we also included ahESC and phESC lines in the analysis.Comparison of DNA methylomes from uniparental blastocysts with those of control blastocysts (produced by intracytoplasmic sperm injection) identified 859 DMRs. Haploid ES cells showed overall erosion of those DMRs, in contrast to diploid ES cells that relatively maintained differential methylation.
Project description:With the increasing use of Assisted Reproductive Technologies (ART) for treatment of human infertility, there is an increasing requirement for embryo culture conditions that perform as similar to nature as possible. How good the match, however, cannot be tested experimentally in human. We solved the central question of how well ART culture protocols prepare embryos for postimplantation development, under the provisions of the 'mouse embryo assay' (MEA). Our side-by-side comparison of 8 conditions [i.e., 3 culture conditions (KSOM, HTF and ISDM1) plus the in vivo system in two different mouse strains (B6 and CD1)] shows that mouse embryos cultured under ART conditions are differentially primed for postimplantation development, and that certain ART protocols outperform the oviduct. The distinct performances of blastocysts formed in ART vs. oviduct do not correlate with any significant transcriptome changes, whereas protein analysis by immunoconfocal microscopy reveals differences in the allocation of embryonic cells to the three germ layers of blastocysts. We conclude that in vitro technology is not always a defective copy of nature, and that the choice of ART protocol primes the embryos for subsequent development. 22 samples were analyzed. B6KSOM: Mouse B6 background, E3.5 blastocysts in KSOM medium, 3 biological rep B6HTF: Mouse B6 background, E3.5 blastocysts in HTF medium, 3 biological rep B6ISM1: Mouse B6 background, E3.5 blastocysts in ISM1 medium, 3 biological rep B6vivo: Mouse B6 background, E3.5 blastocysts in vivo, 3 biological rep CD1KSOM: Mouse CD1 background, E3.5 blastocysts in KSOM medium, 1 biological rep CD1HTF: Mouse CD1 background, E3.5 blastocysts in HTF medium, 3 biological rep CD1ISM1: Mouse CD1 background, E3.5 blastocysts in ISM1 medium, 3 biological rep CD1vivo: Mouse CD1 background, E3.5 blastocysts in vivo, 3 biological rep
Project description:We use MeDIP-sequencing to measure global DNA methylation (5mC) in the uteri of young and aged C57BL/6 mice at day 3.5 (E3.5) of pregnancy, and of young and aged decidua at day 11.5 (E11.5) of pregnancy
Project description:We explored the role of the lysine-methyltrasferase G9a in the control of DNA methylation during mouse embryogenesis. We provide maps of cytosine methylation by MeDIP and RRBS in G9a-deficient mouse embryos and ES cells, as well as ChIP-Seq profiles for H3K9me2 in embryos and RNA-Seq expression profiles in G9a-deficient embryos.
Project description:We explored the role of the lysine-methyltrasferase G9a in the control of DNA methylation during mouse embryogenesis. We provide maps of cytosine methylation by MeDIP and RRBS in G9a-deficient mouse embryos and ES cells, as well as ChIP-Seq profiles for H3K9me2 in embryos and RNA-Seq expression profiles in G9a-deficient embryos.
Project description:In early embryos, DNA methylation is remodelled to initiate the developmental program. For mostly unknown reasons, methylation marks are acquired unequally between embryonic and placental cells. To better understand this, we generated high-resolution maps of DNA methylation in mouse mid-gestation (E10.5) embryo and placenta. We uncovered specific subtypes of differentially methylated regions (DMRs) that contribute directly to the developmental asymmetry existing between mid-gestation embryo and placenta. We show that the asymmetry between embryonic and placental DNA methylation patterns occurs rapidly during the acquisition of marks in the post-implanted conceptus (E3.5-E6.5), and that these patterns are long-lasting across subtypes of DMRs throughout prenatal development and in somatic tissues. We reveal that at the peri-implantation stages, the de novo methyltransferase activity of DNMT3B is the main driver of methylation marks on asymmetric DMRs, and that DNMT3B can largely compensate for lack of DNMT3A in the epiblast and extraembryonic ectoderm, whereas DNMT3A can only partially palliate for the absence of DNMT3B. However, as development progresses and as DNMT3A becomes the principal de novo methyltransferase, the compensatory DNA methylation mechanism on DMRs becomes less effective.
Project description:To characterized the changes in gene expression during the differentiation of TS cells. TS cells can be derived from two time point during embryogenesis, cell lines tested were from each of these time points. Keywords: time course TS cells derived from either E3.5 or E6.5 embryos were differentiated over 6 days by withdrawal of FGF4 and feeder conditioned medium. Cell were sampled every 24 hrs and assayed by microarray.