scholarly journals Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

2021 ◽  
Author(s):  
Masahito Yoshihara ◽  
Ida Kirjanov ◽  
Sonja Nykänen ◽  
Joonas Sokka ◽  
Jere Weltner ◽  
...  
Keyword(s):  
Human Embryo ◽  
Embryonic Stem ◽  
Cell Stage ◽  
Cellular Models ◽  
Transcriptional Dynamics ◽  
Embryonic Genome ◽  
Experimental Approaches ◽  
Genome Activation ◽  
Expression Program

AbstractDUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent DUX4 expression in cellular models. We report here that a transient DUX4 expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

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 Modelling human zygotic genome activation in 8C-like cells in vitro

2021 ◽  
Author(s):  
Jasmin Taubenschmid-Stowers ◽  
Maria Rostovskaya ◽  
Fatima Santos ◽  
Sebastian Ljung ◽  
Ricard Argelaguet ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Genome Activation

The remodelling of the epigenome and transcriptome of the fertilised oocyte to establish totipotency in the zygote and developing embryo is one of the most critical processes in mammalian embryogenesis. Zygotic or embryonic genome activation (ZGA, EGA) in the 2-cell embryo in mouse, and the 8-cell embryo in humans, constitutes the first major wave of transcription. Failure to initiate ZGA leads to developmental defects, and contributes to the high attrition rates of human pre-implantation embryos. Due to limitations in cell numbers and experimental tractability, the mechanisms that regulate human embryonic genome activation in the totipotent embryo remain poorly understood. Here we report the discovery of human 8-cell like cells (8CLCs) specifically among naive embryonic stem cells, but not primed pluripotent cells. 8CLCs express ZGA marker genes such as ZSCAN4, LEUTX and DUXA and their transcriptome closely resembles that of the 8-cell human embryo. 8-cell like cells reactivate 8-cell stage specific transposable elements such as HERVL and MLT2A1 and are characterized by upregulation of the DNA methylation regulator DPPA3. 8CLCs show reduced SOX2 protein, and can be identified based on expression of the novel ZGA-associated protein markers TPRX1 and H3.Y in vitro. Overexpression of the transcription factor DUX4 as well as spliceosome inhibition increase ZGA-like transcription and enhance TPRX1+ 8CLCs formation. Excitingly, the in vitro identified 8CLC marker proteins TPRX1 and H3.Y are also expressed in 8-cell human embryos at the time of genome activation and may thus be relevant in vivo. The discovery of 8CLCs provides a unique opportunity to model and manipulate human ZGA-like transcriptional programs in vitro, and might provide critical functional insights into one of the earliest events in human embryogenesis in vivo.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 113-122 ◽  
Author(s):  
E. Christians ◽  
E. Campion ◽  
E.M. Thompson ◽  
J.P. Renard
Keyword(s):  
Dna Replication ◽  
Mouse Embryo ◽  
Culture Conditions ◽  
Hsp 70 ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Alpha Amanitin ◽  
Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

scholarly journals Human embryonic genome activation initiates at the one-cell stage

2021 ◽  
Author(s):  
Maki Asami ◽  
Brian Y.H. Lam ◽  
Marcella K. Ma ◽  
Kara Rainbow ◽  
Stefanie Braun ◽  
...  
Keyword(s):  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
The One ◽  
Genome Activation

scholarly journals Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 453-465 ◽  
Author(s):  
Irina Lagutina ◽  
Valeri Zakhartchenko ◽  
Helena Fulka ◽  
Silvia Colleoni ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
De Novo ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Cell Stage ◽  
Whole Process ◽  
Embryonic Genome ◽  
Donor Nucleus ◽  
Donor Cells ◽  
Genome Activation

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

69 GLOBAL H3k9ac AND H3k27me3 EXPRESSION IN BLASTOMERES FROM 8- TO 16-CELL STAGE BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 148
Author(s):  
C. S. Oliveira ◽  
N. Z. Saraiva ◽  
L. Z. Oliveira ◽  
R. V. Serapião ◽  
M. R. de Lima ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Histone Modifications ◽  
Pearson Correlation ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Group A ◽  
Genome Activation

Embryonic genome activation is a crucial step in early embryo development, and is accompanied by a dramatic change in the epigenetic profile of blastomeres. Histone modifications related to euchromatin and heterochromatin can be important parameters to infer developmental competence, as they are affected by manipulation and environmental stress conditions. The aim of this study was to characterise permissive (H3k9ac) and repressive (H3k27me3) histone modifications during the embryonic genome activation cell cycle in bovine embryos, regarding correlation between those marks and variance among blastomeres. For that, bovine embryos were produced by IVF and cultured in SOF medium supplemented with 5 mg mL–1 of BSA and 2.5% FCS in 5% O2 in an air atmosphere for 5 days (70 h after IVF). The 8 to 16 cell embryos were fixed in 4% paraformaldehyde and submitted to H3k9ac and H3k27me3 immunofluorescence assay (mouse anti-H3K9ac monoclonal antibody, 1 : 200; Sigma; rabbit anti-H3k27me3 monoclonal antibody, 1 : 200; Upstate, Charlottesville, VA, USA). Nuclei were counterstained with Hoechst 33342. Images of each embryo were captured (AxioCam, Carl Zeiss, São Paulo, Brazil) and measured for nuclear fluorescence intensity in each blastomere using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA, USA). Mean levels were compared using the Mann-Whitney test and variances were compared using F-test (SAS 9.1, SAS Institute Inc., Cary, NC, USA; P = 0.05). We evaluated 2 replicates and 12 embryos during the transition from the 8 to 16 cell stages, totaling 169 blastomeres. Global H3k27me3 levels varied accordingly to H3k9ac levels, as indicated by a high Pearson correlation coefficient (r = 0.913). Levels of each blastomere were normalized to the lowest level obtained within each embryo. Some embryos displayed a high variation between blastomeres, and, for further analysis, we divided the embryos into groups: group A for embryos that presented similar H3k9ac levels between blastomeres (8 embryos, 66%), and group B for embryos that exhibited higher heterogeneity between blastomeres (at least 2 blastomeres presenting a 2-fold increase compared to the lowest blastomere; 4 embryos, 33%). Mean H3k9ac and H3k27me3 normalized levels were lower for group A [H3k9ac: 1.35 ± 0.29 (A), 1.94 ± 1.02* (B); H3k27me3: 1.33 ± 0.24 (A), 1.99 ± 0.77 (B)], and group A displayed lower variance values (H3k9ac: 0.07 (A), 1.05* (B); H3k27me3: 0.06 (A), 0.60 (B)]. Within each embryo, blastomeres were sorted in ascending order for H3k9ac level (1 to 16), and compared between groups A and B. We detected that mean levels differed (P < 0.05) between groups from blastomere 9 to 16 for H3k9ac and 10 to 16 for H3k27me3. Therefore, in 8- to 16-cell stage embryos, the H3k27me3 repressive mark is highly correlated with the H3k9ac permissive mark. Also, our results describe the presence of 2 distinguishable populations of bovine embryos at this stage, considering their epigenetic status. One population presented similar levels of repressive and permissive marks among blastomeres, whereas the second one displayed a remarkable variation among their blastomeres. This observation should be further studied, as it might reflect distinct cleavage pattern embryos and blastomere competence. The authors acknowledge FAPESP, FAPERJ and CNPq for financial support.

61 REVERSIBLE INHIBITION OF BOVINE MINOR EMBRYONIC GENOME ACTIVATION IMPAIRS PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 138
Author(s):  
R. P. Nociti ◽  
R. V. Sampaio ◽  
V. F. M. H. de Lima ◽  
R. M. Schultz ◽  
P. J. Ross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reversible Inhibitor ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome Activation ◽  
Transcriptional Inhibition ◽  
Embryonic Genome ◽  
Genome Activation

Bovine pre-implantation embryos can develop in the absence of gene expression up to the 8/16-cell stage, the time when the major embryonic genome activation (EGA) occurs. Some embryonic genes, however, are transcribed before EGA (minor EGA). This study used a reversible inhibitor of RNA Polymerase II (5,6 dichlorobenzimidazole 1-β-D-ribofuranoside; DRB) to assess the importance of minor EGA for development to the blastocyst stage. Oocytes were matured and inseminated in vitro, and the fertilized eggs were cultured in supplemented KSOMaa and allocated to different treatments 16 h post-insemination (hpi). Development was recorded at 44 and 72 hpi, and the incidence of blastocyst formation on Day 7 (IVF = Day 0) was recorded. Data were analysed by ANOVA followed by Duncan test. First, we tested different DRB concentrations [50 μM (D50), 75 μM (D75), 100 μM (D100), and dimethyl sulfoxide vehicle control (CTRL)] to block development to blastocyst when continuously present. Only embryos in CTRL produced blastocysts (45.0 ± 5.8%; 4 replicates with a total of 391 oocytes examined). No difference in development was observed at 44 h (57.9 ± 16.5, 53.3 ± 10.5, 60.5 ± 19.0, and 52.3 ± 5.8% for D50, D75, D100, and CTRL, respectively) and 72 h (78.9 ± 8.8, 66.1 ± 11.7, 71.5 ± 16.5, and 70.8 ± 5.6% for D50, D75, D100, and CTRL, respectively). Next, in 7 replicates (751 oocytes) we determined the effect of blocking transcription (50 μM DRB) spanning 2 embryo stages (periods of 28 h), initiated at 16 hpi (1&2C), 30 hpi (2&4C), and 44 hpi (4&8C). Controls included DRB treatment from 16 to 72 hpi (1–8C) and CTRL. There was no difference in development at 44 and 72 h. The incidence of blastocyst formation, however, was significantly decreased in all treatment groups compared with CTRL (27.7 ± 4.7; 15.1 ± 3.5; 23.3 ± 3.1; 20.5 ± 1.9; and 42.1 ± 3.2% for 1&2C, 2&4C, 4&8C, 1–8C, and CTRL, respectively). Finally, in 12 replicates (1499 oocytes), the effect of blocking transcription for 14-h periods, spanning mostly a unique cleavage stage, was evaluated. The DRB treatment (50 μM) started at 16 hpi (1C), 30 hpi (2C), 44 hpi (4C), and 58 hpi (8C). Furthermore, 1–16C and CTRL treatments were included. No difference in development at 44 and 72 h were observed. Development to the blastocyst was significantly lower from CTRL (46.0 ± 3.2%) in 2C, 4C, 8C, and 1–16C (28.9 ± 3.9, 26.1 ± 4.2, 30.1 ± 4.8, and 18.9 ± 3.2%, respectively) but not in 1C (34.7 ± 4.4%). In summary, continuous transcriptional inhibition using DRB resulted in a developmental block at the time of major EGA, similar to α-amanitin treatment (an irreversible RNA Polymerase II inhibitor). Transcriptional inhibition during single cleavage stages was sufficient to decrease the developmental potential of the embryo. We conclude that minor EGA has an important role for bovine development. This work was funded by NIH-NICHD R01HD070044 to P. J. Ross. R. P. Nociti was sponsored by CNPQ; R. V. Sampaio was sponsored by FAPESP.

45 Expression patterns of PRDM family genes in porcine pre-implantation embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 148
Author(s):  
K. Farrell ◽  
K. Uh ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Internal Control ◽  
Target Genes ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Cell Stage ◽  
Early Embryo Development

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values&lt;0.05 were considered significant. There was a significant decrease in PRDM2 expression in 4-cell and blastocyst, PRDM4 expression in 4-cell, and PRDM6 in all stages (MII, zygote, 4-cell, and blastocyst), compared with the GV stage. Because zygotic genome activation occurs at the 4-cell stage in the pig, the significant decrease in gene expression (PRDM2, PRDM4, and PRDM6) indicates they may be maternally originated and involved in the reprogramming process following fertilisation. On the other hand, there was a significant increase in PRDM15 expression in blastocysts and the PRDM14 transcript was only detected in blastocysts in all three biological replicates, suggesting that the genes are most likely involved in pluripotency maintenance, as was found in previous human studies. These results indicate that PRDM family genes are differentially expressed during early embryo development in pigs and may play a role in maintenance of pluripotency. For further study, we intend to evaluate the role of PRDM family genes during early embryo development in pigs.

scholarly journals A transcriptional roadmap for 2C-like–to–pluripotent state transition

2020 ◽  
Vol 6 (22) ◽  
pp. eaay5181 ◽  
Author(s):  
Xudong Fu ◽  
Mohamed Nadhir Djekidel ◽  
Yi Zhang
Keyword(s):  
Intermediate State ◽  
State Transition ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Small Cell ◽  
Molecular Event ◽  
Cell Stage ◽  
Pluripotent State ◽  
Transcriptional Dynamics ◽  
The One

In mouse embryonic stem cell (ESC), a small cell population displays totipotent features by expressing a set of genes that are transiently active in 2-cell–stage embryos. These 2-cell–like (2C-like) cells spontaneously transit back into the pluripotent state. We previously dissected the transcriptional dynamics of the transition from pluripotency to the totipotent 2C-like state and identified factors that modulate the process. However, how 2C-like cells transit back into the pluripotent state remains largely unknown. In this study, we analyzed the transcriptional dynamics from the 2C-like state to pluripotent ESCs and identified an intermediate state. The intermediate state characterized by two-wave step up-regulation of pluripotent genes is different from the one observed during the 2C-like entry transition. Nonsense-mediated Dux mRNA decay plays an important role in the 2C-like state exit. Thus, our study not only provides a transcriptional roadmap for 2C-like–to–pluripotent state transition but also reveals a key molecular event driving the transition.

scholarly journals The role of ZFP57 and additional KRAB-zinc finger proteins in the maintenance of human imprinted methylation and multi-locus imprinting disturbances

2020 ◽  
Vol 48 (20) ◽  
pp. 11394-11407
Author(s):  
Ana Monteagudo-Sánchez ◽  
Jose Ramon Hernandez Mora ◽  
Carlos Simon ◽  
Adam Burton ◽  
Jair Tenorio ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Expression Profiling ◽  
Zinc Finger Proteins ◽  
Early Embryogenesis ◽  
Mouse Embryos ◽  
Parental Origin ◽  
Embryonic Genome ◽  
Repressor Complex ◽  
Genome Activation

Abstract Genomic imprinting is an epigenetic process regulated by germline-derived DNA methylation that is resistant to embryonic reprogramming, resulting in parental origin-specific monoallelic gene expression. A subset of individuals affected by imprinting disorders (IDs) displays multi-locus imprinting disturbances (MLID), which may result from aberrant establishment of imprinted differentially methylated regions (DMRs) in gametes or their maintenance in early embryogenesis. Here we investigated the extent of MLID in a family harbouring a ZFP57 truncating variant and characterize the interactions between human ZFP57 and the KAP1 co-repressor complex. By ectopically targeting ZFP57 to reprogrammed loci in mouse embryos using a dCas9 approach, we confirm that ZFP57 recruitment is sufficient to protect oocyte-derived methylation from reprogramming. Expression profiling in human pre-implantation embryos and oocytes reveals that unlike in mice, ZFP57 is only expressed following embryonic-genome activation, implying that other KRAB-zinc finger proteins (KZNFs) recruit KAP1 prior to blastocyst formation. Furthermore, we uncover ZNF202 and ZNF445 as additional KZNFs likely to recruit KAP1 to imprinted loci during reprogramming in the absence of ZFP57. Together, these data confirm the perplexing link between KZFPs and imprint maintenance and highlight the differences between mouse and humans in this respect.

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