EZH2 is essential for development of mouse preimplantation embryos

2014 ◽  
Vol 26 (8) ◽  
pp. 1166 ◽  
Author(s):  
Xian-Ju Huang ◽  
Xuguang Wang ◽  
Xueshan Ma ◽  
Shao-Chen Sun ◽  
Xiaolong Zhou ◽  
...  
Keyword(s):  
De Novo ◽  
Preimplantation Embryo ◽  
Epigenetic Modification ◽  
Es Cells ◽  
Embryonic Stem ◽  
Giant Cells ◽  
Preimplantation Embryos ◽  
Preimplantation Embryo Development ◽  
Expression Of Genes ◽  
Early Mouse

Enhancer of zeste homologue 2 (Ezh2) is essential for the development of the early mouse preimplantation embryo. Loss of Ezh2 results in embryonic lethality in mice. Ezh2-deficient embryos display impaired outgrowth potential, defective establishment of Ezh2-null embryonic stem (ES) cells and adherence and differentiation of the trophoblast layer into giant cells. We investigated if Ezh2 controls the fate of embryos at an earlier stage by treating with cycloheximide (CHX) or microinjecting short interfering RNA (siRNA) to restrict embryonic Ezh2 expression during preimplantation. CHX inhibited de novo EZH2 protein synthesis in zygotes, suggesting that EZH2 requires de novo synthesis during post-fertilisation stages. We found that loss of Ezh2 at the pronuclear stage caused severe growth retardation and reduced blastocyst formation. Expression of the pluripotency-associated markers Oct4, Sox2 and Nanog were significantly decreased in embryos that had been injected with Ezh2 siRNA. In addition, Ezh2 loss induced upregulated expression of genes related to the differentiation of germ layers, including Gata6, Hoxb1 and Hand1. Finally, apoptosis was increased in the blastocyst embryos with Ezh2 knockdown. Modification of histone H3-Lysine 27 de-methylation and tri-methylation (H3K27me2/3) was strongly reduced in Ezh2 siRNA embryos. We conclude that Ezh2 is essential for early preimplantation embryo development through the regulation of epigenetic modification and apoptosis.

scholarly journals Differential Recruitment of Methylated CpG Binding Domains by the Orphan Receptor GCNF Initiates the Repression and Silencing of Oct4 Expression

2006 ◽  
Vol 26 (24) ◽  
pp. 9471-9483 ◽  
Author(s):  
Peili Gu ◽  
Damien Le Menuet ◽  
Arthur C.-K. Chung ◽  
Austin J. Cooney
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Epigenetic Modification ◽  
Es Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Orphan Receptor ◽  
Proximal Promoter ◽  
Es Cell ◽  
Oct4 Expression

ABSTRACT The pluripotent factor Oct4 is a key transcription factor that maintains embryonic stem (ES) cell self-renewal and is down-regulated upon the differentiation of ES cells and silenced in somatic cells. A combination of cis elements, transcription factors, and epigenetic modifications, such as DNA methylation, are involved in the regulation of Oct4 gene expression. Here we show that the orphan nuclear receptor GCNF initiates Oct4 repression and DNA methylation by the differential recruitment of MBD (methylated CpG binding domain) factors to the promoter. Compared with wild-type ES cells and gastrulating embryos, Oct4 repression is lost and its proximal promoter is significantly hypomethylated in RA-differentiated GCNF−/− ES cells. The Oct4 gene is reexpressed in some somatic cells of GCNF−/− embryos, showing that it has not been properly silenced coincident with reduced DNA methylation of its promoter. Efforts to characterize mediators of GCNF's repressive function and DNA methylation of the Oct4 promoter identified methyl-DNA binding proteins, MBD3 and MBD2, as GCNF-interacting factors. In P19 and ES cells, upon differentiation, endogenous GCNF binds to the Oct4 proximal promoter and differentially recruits MBD3 and MBD2. In differentiated GCNF−/− ES cells, recruitment of MBD3 and MBD2 to the Oct4 promoter is lost, and repression of Oct4 expression and DNA methylation fails to occur. RNA interference-mediated knockdown of MBD3 and/or MBD2 expression results in reduced Oct4 repression in differentiated P19 and ES cells. Repression of Oct4 expression and recruitment of MBD3 are maintained in de novo DNA methylation-deficient ES cells (Dnmt3A/3B-null cells), while MBD2 recruitment is lost. Thus, recruitment of MBD3 and MBD2 by GCNF links two events, gene-specific repression and DNA methylation, which occur differentially at the Oct4 promoter. GCNF initiates the repression and epigenetic modification of Oct4 gene during ES cell differentiation.

scholarly journals Identification and differential expression patterns of porcine OCT4 variants

Reproduction ◽  
2015 ◽  
Vol 149 (1) ◽  
pp. 55-66 ◽  
Author(s):  
Jae Yeon Hwang ◽  
Jong-Nam Oh ◽  
Dong-Kyung Lee ◽  
Kwang-Hwan Choi ◽  
Chi-Hun Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Crucial Role ◽  
Preimplantation Embryo ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Preimplantation Embryo Development ◽  
Cryptic Exon

OCT4 encoded by POU5F1 has a crucial role of maintaining pluripotency in embryonic stem cells during early embryonic development and several OCT4 variants have been identified in mouse and human studies. The objective of this study was to identify different variants of OCT4 and analyze their expression patterns in preimplantation porcine embryos and various tissues. In this study, we showed that POU5F1 transcribes its three variants, namely OCT4A, OCT4B, and OCT4B1. The OCT4B transcript consists of exons identical to the major form of the OCT4 variant, OCT4A, with a differential N-terminal domain-coding exon. The structure of OCT4B1 mRNA was the same as that of OCT4B mRNA, but harbored a cryptic exon. Based on these findings, the transcription levels were investigated and found that OCT4B and OCT4B1 made up ∼20% among the variants in the embryonic stage and this indicates that OCT4A mRNA is dominantly expressed during preimplantation embryo development. In addition, OCT4B mRNA was detected in all tissues examined, while OCT4A and OCT4B1 were detected only in testis but not in other tissues examined. OCT4B1 showed inversely correlated expression with SOX2 and NANOG expression. OCT4A protein was specifically localized to the nuclei, whereas OCT4B was mainly localized to the cytoplasm of the porcine embryos at the blastocyst stage. The findings of this study reveal that the porcine OCT4 gene can potentially encode three variants (OCT4A, OCT4B, and OCT4B1), and they are differentially expressed and would have roles dissimilar between each other in preimplantation embryos and various adult tissues.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

scholarly journals Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice

2021 ◽  
Vol 9 ◽  
Author(s):  
Yuanyuan Li ◽  
Ning-Hua Mei ◽  
Gui-Ping Cheng ◽  
Jing Yang ◽  
Li-Quan Zhou
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Zygotic Genome Activation ◽  
Preimplantation Embryo Development ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Genome Activation ◽  
Zygotic Genome

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.

258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

2008 ◽  
Vol 20 (9) ◽  
pp. 58
Author(s):  
G. R. Kafer ◽  
SA Lehnert ◽  
P. L. Kaye ◽  
R. J. Moser
Keyword(s):  
Messenger Rna ◽  
Expression Profiles ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Histone Variants ◽  
Histone Variant ◽  
Preimplantation Embryos ◽  
Es Cell

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.

scholarly journals Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Elo Madissoon ◽  
Eeva-Mari Jouhilahti ◽  
Liselotte Vesterlund ◽  
Virpi Töhönen ◽  
Kaarel Krjutškov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Target Genes ◽  
Genomic Sequence ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Preimplantation Embryo Development ◽  
Genome Activation

Abstract PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.

The chicken stathmin gene and its expression in the embryo

2000 ◽  
Vol 78 (6) ◽  
pp. 703-713 ◽  
Author(s):  
Sharon Soodeen-Karamath ◽  
Ann M Verrinder Gibbins
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Early Mouse Embryo ◽  
Differentiated Cells ◽  
Selective Ablation ◽  
Potential Indicator ◽  
Early Mouse ◽  
Intron 2

Stathmin, which functions as an intracellular relay in signal transduction pathways, has been suggested as a potential indicator of pluripotent cells in the early mouse embryo. In this study, chicken stathmin cDNA and genomic DNA were analyzed. In mammals stathmin consists of five exons and four introns; exons 3, 4, and 5 in the mammalian stathmin gene are equivalent to one relatively large exon in the chicken stathmin gene. Introns equivalent to introns 3 and 4 in the mammalian stathmin gene are not present in the counterpart gene in chickens and, although intron 2 was shown to be present in both mammals and birds, it is smaller in the chicken stathmin gene. Despite differences in the genomic organization of the gene and its smaller size in chickens compared with that in humans and mice, similarities in the coding sequences and in the expression of the chicken and mouse stathmin genes at certain stages of embryo development, as determined by whole-mount in situ hybridization experiments, suggest that their products are functional homologues. The argument is thus substantiated for further investigations into the use of regulatory regions of the stathmin gene in a system for the establishment of long-term cultures of germline competent chicken embryonic stem (ES) cells by the selective ablation of differentiated cells in culture using drug selection.Key words: stathmin, chicken, ES cells, oct 3/4.

scholarly journals Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells

2015 ◽  
Vol 47-48 ◽  
pp. 101-109 ◽  
Author(s):  
Silvia Muñoz-Descalzo ◽  
Anna-Katerina Hadjantonakis ◽  
Alfonso Martinez Arias
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Early Mouse

scholarly journals Phosphatidylinositol 3-kinase signaling in mammalian preimplantation embryo development

Reproduction ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 147-156 ◽  
Author(s):  
Chris O'Neill
Keyword(s):  
Embryo Development ◽  
Normal Development ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
External Information ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Preimplantation Embryo Development ◽  
Wide Range ◽  
Phosphatidylinositol 3

The development of the preimplantation mammalian embryo is an autopoietic process; once initiated development proceeds without an absolute requirement for external information or growth cues. This developmental autonomy is partly explained by the generation of autocrine trophic ligands that are released and act back on the embryo via specific receptors. Several embryotrophic ligands cause receptor-dependent activation of 1-o-phosphatidylinositol 3-kinase. This enzyme phosphorylates phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate. Genetic or pharmacological ablation of this enzyme activity disrupts normal development of preimplantation embryos. Phosphatidylinositol-3,4,5-trisphosphate is a membrane lipid that acts as a docking site for a wide range of proteins possessing the pleckstrin homology (PH) domain. Such proteins are important regulators of cell survival, proliferation, and differentiation. RAC-α serine/threonine protein kinase is an important PH domain protein and its activity is required for normal preimplantation embryo development and survival. The activity of a range of PH domain proteins is also implicated in the normal development of the embryo. This review critically examines the evidence for the activation of 1-o-phosphatidylinositol 3-kinase in the generation of pleiotypic trophic response to embryotrophins in the autopoietic development of the preimplantation embryo.

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