Identification of Oct4-dependent genes in vivo
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE47089 | GEO | 2013/06/06
SECONDARY ACCESSION(S): PRJNA203536
REPOSITORIES: GEO
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Project description:Transcriptome analysis of Oct4 null and wild type preimplantation mouse embryos by RNA-sequencing N=3 embryos of two genotypes (+/+ or -/-) at each of three time points (E2.5, E3.75, E4.5) were sequenced.
2013-06-06 | E-GEOD-47089 | biostudies-arrayexpress
Project description:OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signalling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, upregulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.
2021-01-03 | GSE159030 | GEO
Project description:The goal is to examine the transcriptome of Oct4 and Nanog conditional expressing mouse embryos at different developmental stages.
2020-01-01 | GSE94952 | GEO
Project description:During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 directly activate the pluripotency-related genes through the corresponding OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory towards the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.
2024-10-14 | GSE264615 | GEO
Project description:During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 directly activate the pluripotency-related genes through the corresponding OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory towards the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.
2024-10-14 | GSE264614 | GEO
Project description:OCT4 was knocked down doxycycline-dependently, and then wild type or S236 phosphorylation mimicking OCT4 were rescued.
2020-07-01 | GSE117521 | GEO
Project description:Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR?Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocys t development was established, but maintenance was compromised. We conclude that CRISPR?Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
2017-09-21 | GSE100119 | GEO
Project description:We set out to develop an unbiased, genome-wide method to screen for proteins that locate to the OCT4 proximal enhancer (PE) in hESCs; for this purpose, we developed an optimized locus-specific proteomics approach in hESCs. First, we designed TALEN (Transcription Activator-Like Effector Nuclease) constructs to target the sequences that are near the OCT4 PE, located in a region of DNaseI hypersensitivity. TALEN constructs with the highest cutting efficiency were chosen for locus-specific proteomics. We then made modifications to the original TALEN protein to transform it into a catalytically-dead TALE (dTALE) protein that is optimized for locus-specific proteomics in hESCs via three steps: 1) The nuclease-domain FokI at the C-terminus was replaced by a GFP (green fluorescence protein); 2) a 3X FLAG tag at the N-terminus was included for following pull-down analysis; 3) the existing CMV promoter was replaced with an EF1alpha promoter that has robust expression in hESCs. This dTALE protein could then be chemically crosslinked to the OCT4 locus together with all the other proteins that bind to the locus. We verified that dTALE protein binds to the targeted locus by chromatin immunoprecipitation (ChIP)-qPCR. Following crosslinking, chromatin was sheared, and all the associated proteins were immunoprecipitated using an anti-FLAG antibody. Immunoprecipitation pulled down the dTALE protein as well as other proteins and complexes that are also attached to that region. Crosslinking was then reversed, and the samples were subjected to mass spectrometry to enable generation of a list of proteins that potentially bind to OCT4 PE locus.
2019-01-07 | PXD010963 | Pride
Project description:Gene regulation at the maternal-embryonic transition in the pre-implantation mouse embryo is not well understood. We knock down Ccna2 to establish proof-of-concept that antisense morpholino oligonucleotides can be used to target specific genes. We applied this strategy to study Oct4 and discovered that Oct4 is required prior to blastocyst development. Specifically, gene expression is altered as early as the 2-cell stage in Oct4-knockdown embryos. Distinct subsets of genes are differentially expressed between Oct4 and Ccna2-knockdown embryos, and indicated differential functions. Further, a large panel of genes were confirmed to be differentially-expressed in Oct4-knockdown embryos by quantitative, real time RT-PCR. Keywords: gene knockdown 3-5 week old wild type F1 (C57BL6xDBA/2) females (Charles River) were superovulated by intraperitonial injections of 5 IU of pregnant mare’s serum gonadotropin (Sigma) followed by 5 IU of human chorionic gonadotropin (Sigma) 48 hours later, and mated overnight with wild type males. Mice were sacrificed by cervical dislocation 17 hours after hCG injection, and 1-cell embryos were released from oviducts. Cumulus cells were removed by hyaluronidase (Sigma) treatment and pipetting. Pre-implantation embryos at the two pronuclei stage were recovered, pooled from 3-6 females in M2 media (Chemicon International), followed by immediate cytoplasmic microinjection of gene-specific antisense morpholino oligonucleotides and culture in Human Tubal Fluid with 10% serum supplement (In-Vitro Fertilization, Inc.) microdrops under mineral oil (Sigma) in mixed gas (90% nitrogen, 5% oxygen, 5% carbon dioxide; Praxair) at 37°C, and cultured at ten embryos per 20 μL drop. Uninjected control embryos derived from the same embryo pool, and were placed in identical conditions in parallel, except that they were not injected.
2008-12-30 | E-GEOD-13981 | biostudies-arrayexpress
Project description:Gene regulation at the maternal-embryonic transition in the pre-implantation mouse embryo is not well understood. We knock down Ccna2 to establish proof-of-concept that antisense morpholino oligonucleotides can be used to target specific genes. We applied this strategy to study Oct4 and discovered that Oct4 is required prior to blastocyst development. Specifically, gene expression is altered as early as the 2-cell stage in Oct4-knockdown embryos. Distinct subsets of genes are differentially expressed between Oct4 and Ccna2-knockdown embryos, and indicated differential functions. Further, a large panel of genes were confirmed to be differentially-expressed in Oct4-knockdown embryos by quantitative, real time RT-PCR. Keywords: gene knockdown
2008-12-31 | GSE13981 | GEO