Project description:Primordial germ cell mRNA profiles from cells microdissected from e6.5, e7.5 and e8.5 embryos, e7.5 somatic neighbours and Blimp1-KO mice were generated by single cell library construction and sequencing in duplicate using Applied Biosystems SOLiD sequencer. Single cell library construction is described in: Tang f. et. al, Nature Protocols (2010), Vol. 5, p.516.
Project description:Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers. Useful links: Parsed data: (ftp://ftp.ebi.ac.uk/pub/databases/scnmt_gastrulation) Github repository: (https://github.com/rargelaguet/scnmt_gastrulation)
Project description:Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers. Useful links: Parsed data: (ftp://ftp.ebi.ac.uk/pub/databases/scnmt_gastrulation) Github repository: (https://github.com/rargelaguet/scnmt_gastrulation)
Project description:Transcription profiling by high throughput sequencing of primordial germ cells from e6.5, e7.5, e8.5 embryos and e7.5 somatic neighbours and Blimp1-KO mice
Project description:Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng+Flk1+ mesodermal precursor population at E7.5, a cell fraction also endowed with endothelial potential. In Eng knockout embryos, hematopoietic colony activity and numbers of CD71+Ter119+ erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key BMP target genes including the hematopoietic regulators Scl, Gata1, Gata2 and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng+Flk-1+ progenitors co-expressed TGFβ and BMP receptors and target genes. Furthermore, Eng+Flk-1+ cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of endoglin-expressing cells was dependent on TGFβ superfamily ligands: BMP4, BMP2, or TGF-β1. These data demonstrate that the E+F+ fraction at E7.5 represents mesodermal cells competent to respond to TGFb1, BMP4, or BMP2, shaping their hematopoietic development, and that endoglin is a critical regulator in this process by modulating TGF/BMP signaling. E7.5 pooled embryos (25 litters; 300 embryos approximately) were dissected and 3,000 cells were sorted in triplicate for Eng-Flk1-, Eng-Flk1+, Eng+Flk1+, and Eng+Flk1- fractions. Microarray results were analyzed with GeneSpring GX software.
Project description:The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization (IVF) were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (> 2-fold vs. controls) than did the extraembryonic tissues (P < 1.0 M-CM-^W 10M-bM-^@M-^S26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1M-bM-^@M-^S5% per embryo transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages, although these alterations were not always statistically significant for all three SCNT groups because of variability. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. Comparative gene expression analyses using post-implanted E6.5 cloned embryos were performed by microarray. Cloned embryos were produced with three different types of donor cells (cumulus cells, neonatal Sertoli cells and fibroblasts). As controls, sex- and genotype-matched embryos produced by in vitro fertilization were used. Each embryos were mechanically dissected into the embryonic and extraembryonic parts at E6.5 and were subjected to gene expression microarray.
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.