DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and preimplantation development

How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive. Recent studies indicate that DPPA2 and DPPA4 are required for establishing a 2-cell embryo-like (2C-like) state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that Dux activation, ZGA, and preimplantation development are normal in embryos without DPPA2 or DPPA4. Thus, unlike in ESCs/2C-like cells, DPPA2 and DPPA4 are dispensable for ZGA and preimplantation development.

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DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and preimplantation development

Development ◽

10.1242/dev.200178 ◽

2021 ◽

Author(s):

Zhiyuan Chen ◽

Zhenfei Xie ◽

Yi Zhang

Keyword(s):

De Novo ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Preimplantation Development ◽

Dependent Manner ◽

Maternal Factors ◽

Zygotic Genome Activation ◽

Genome Activation ◽

Zygotic Genome

How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive in mammals, partly due to the challenge of de novo identification of key factors using scarce materials. The 2-cell (2C) embryo like cells has been widely used as an in vitro model to understand mouse ZGA and totipotency given its expression of a group of 2C embryo-specific genes and its simplicity for genetic manipulation. Recent studies indicate that DPPA2 and DPPA4 are required for establishing the 2C-like state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that DPPA2 and DPPA4 are dispensable for Dux activation, ZGA, and preimplantation development. Our study suggests that 2C-like cells do not fully recapitulate 2-cell embryos in terms of 2C-gene regulation and cautions should be taken when studying ZGA and totipotency using 2C-like cells as the model system.

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DPPA2 and DPPA4 are necessary to establish a totipotent state in mouse embryonic stem cells

10.1101/447755 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alberto De Iaco ◽

Alexandre Coudray ◽

Julien Duc ◽

Didier Trono

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Early Embryo ◽

Zygotic Genome Activation ◽

Cell Stage ◽

Oocyte Expression ◽

Genome Activation ◽

Zygotic Genome

AbstractAfter fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through so-called zygotic genome activation (ZGA), occurring in the mouse at the 2-cell stage. Amongst the first elements to be transcribed are the Dux gene, the product of which secondarily induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons, which regulate chromatin accessibility in the early embryo. The maternally-inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Here we identify the paralog proteins DPPA2 and DPPA4 as responsible for this process.

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.788512 ◽

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.

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Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

Development ◽

10.1242/dev.200191 ◽

2021 ◽

Vol 148 (24) ◽

Author(s):

Oana Kubinyecz ◽

Fatima Santos ◽

Deborah Drage ◽

Wolf Reik ◽

Melanie A. Eckersley-Maslin

Keyword(s):

Null Allele ◽

Embryonic Stem ◽

Offspring Survival ◽

Zygotic Genome Activation ◽

Molecular Events ◽

Genome Activation ◽

Mitotic Chromatin ◽

Zygotic Genome

ABSTRACT Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

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Lipid rafts integrity is essential for the prolactin-induced mitogenesis in mouse embryonic stem cells

The International Journal of Developmental Biology ◽

10.1387/ijdb.210194dm ◽

2021 ◽

Author(s):

Sophia Karouzaki ◽

Charoula Peta ◽

Emmanouella Tsirimonaki ◽

George Leondaritis ◽

Kostas Vougas ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Lipid Rafts ◽

Membrane Lipids ◽

Close Association ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Mass Spectrometry Analysis ◽

Signalling Pathways ◽

Dependent Manner

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

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MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

10.1101/2021.08.11.456030 ◽

2021 ◽

Author(s):

Majid Mehravar ◽

Yogesh Kumar ◽

Moshe Olshansky ◽

Dhiru Bansal ◽

Craig Dent ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Biology ◽

Messenger Rna ◽

Mrna Decay ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Beta Catenin ◽

Dependent Manner ◽

Leukaemia Virus

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates, such as splicing, export, localisation, decay, stability, and translation to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in mouse embryonic stem cells (mESCs). MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A- containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3beta mRNA through m6A that stabilises the BETA-CATENIN expression of a WNT/BETA-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mESC state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

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304 THE DEVELOPMENTAL TOXICITY EVALUATION OF 5-FLUOROURACIL AND INDOMETHACIN IN HUMAN EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab304 ◽

2013 ◽

Vol 25 (1) ◽

pp. 299

Author(s):

E. M. Jung ◽

E. B. Jeung

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Developmental Process ◽

Developmental Toxicity ◽

Embryonic Stem ◽

Surface Markers ◽

Dependent Manner ◽

Undifferentiated Hesc

Embryonic stem cells have pluripotency and differentiate into and constitute the cells and tissues of our body. In this study, using human embryonic stem cells (hESC), we evaluated novel methods for screening toxicological chemicals during developmental process. We elucidated developmental toxicity of two well-known chemicals, 5-fluorouracil (5-FU) and indomethacin (Indo) in hESC. The undifferentiated hESC were treated with the chemicals (10–4 to 104 µM of 5-FU and Indo) in a dose-dependent manner during 1 to 3 days. Surface markers (SSEA-4, TRA-1-60, and TRA-1-81) expressed only in undifferentiated hESC were monitored by immunocytochemistry to ensure the characterisation of undifferentiated hESC. Moreover, expression of embryonic stem cell-specific genes was assessed with real-time PCR after treatment of 5-FU and Indo (10–2, 100, and102 µM of 5-FU and Indo). The expression of surface markers was not significantly affected by treatment of 5-FU and Indo. The expression of transcription factors (Oct-4, Sox-2, Nanog, and hTERT) was significantly decreased by high concentrations of 5-FU and Indo (102 µM). However, no difference was observed in treatment of low concentration of 5-FU and Indo (10–2 µM). Taken together, these results suggest that 5-FU and Indo have cytotoxic effects, and modulate the expression of transcription factors that have pivotal roles in undifferentiated hESC. Therefore, we suggest that hESC may have potential to test toxicity of chemicals during embryonic developmental stage.

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278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab278 ◽

2009 ◽

Vol 21 (1) ◽

pp. 236

Author(s):

E.-M. Jeung ◽

K.-C. Choi ◽

E.-B. Jeung

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Embryoid Bodies ◽

Time Dependent ◽

Dependent Manner ◽

Cardiomyocyte Differentiation ◽

Differentiation Markers ◽

Oct4 Expression

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

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