scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Alex Chialastri ◽  
Jean-Charles Boisset ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development

AbstractDNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.

scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2019 ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Jean-Charles Boisset ◽  
Alex Chialastri ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development ◽  
Genome Wide

AbstractDNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

2008 ◽  
Vol 20 (1) ◽  
pp. 109
Author(s):  
O. Svarcova ◽  
A. Dinnyes ◽  
Z. Polgar ◽  
S. Bodo ◽  
M. Adorjan ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Ultrastructural Changes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Stage Of Development ◽  
Nucleolar Proteins ◽  
Genome Activation ◽  
Different Origin

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).

scholarly journals Delayed and stage specific phosphorylation of H2AX during preimplantation development of γ-irradiated mouse embryos

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Satish Kumar Adiga ◽  
Megumi Toyoshima ◽  
Tsutomu Shimura ◽  
Jun Takeda ◽  
Norio Uematsu ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Early Stage ◽  
In Utero ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Γ Irradiation ◽  
Cell Stage ◽  
Focus Formation ◽  
Damage Site ◽  
Stage Of Development

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as γ-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of γ-H2AX in mouse embryos γ-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy γ-irradiation is hindered up to the two cell stage of development. When left in utero for another 24–64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

scholarly journals Retinoic acid signaling is critical during the totipotency window in early mammalian development

2021 ◽  
Author(s):  
Ane Iturbide ◽  
Mayra L. Ruiz Tejeda Segura ◽  
Camille Noll ◽  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Networks ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mammalian Development ◽  
Cell Stage ◽  
Cell Potential ◽  
Cellular Models ◽  
Early Embryos ◽  
Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

scholarly journals Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

2015 ◽  
Vol 112 (31) ◽  
pp. E4236-E4245 ◽  
Author(s):  
Jinsuk Kang ◽  
Matthias Lienhard ◽  
William A. Pastor ◽  
Ashu Chawla ◽  
Mark Novotny ◽  
...  
Keyword(s):  
Single Cells ◽  
Cholesterol Biosynthesis ◽  
Early Embryogenesis ◽  
Dna Demethylation ◽  
Cell Stage ◽  
Tet Proteins ◽  
Genes Encoding ◽  
Variable Gene ◽  
Intracellular Pathways ◽  
Tet Enzymes

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
W. J. D. Reeve ◽  
C. A. Ziomek
Keyword(s):  
Ligand Binding ◽  
Single Cells ◽  
Progressive Increase ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Characteristic Distribution ◽  
Surface Polarization ◽  
Cell Embryo ◽  
Fluorescent Ligand ◽  
Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

scholarly journals The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

2016 ◽  
Vol 39 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Martin Morovic ◽  
Matej Murin ◽  
Frantisek Strejcek ◽  
Michal Benc ◽  
Dusan Paál ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Coming Out ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Early Embryos

AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

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