scholarly journals CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos

Reproduction ◽  
2020 ◽  
Vol 159 (6) ◽  
pp. 767-778
Author(s):  
Virginia Savy ◽  
Virgilia Alberio ◽  
Natalia G Canel ◽  
Laura D Ratner ◽  
Maria I Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Embryo Development ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Mammalian Embryos

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 155
Author(s):  
V. Savy ◽  
V. Alberio ◽  
N. Canel ◽  
L. Ratner ◽  
M. Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Regulatory Region ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Guide Rnas ◽  
Endogenous Expression ◽  
Mammalian Embryos

The CRISPR-Cas9 system enables precise genome editing in mammalian somatic cells and embryos at a very high efficiency. A modified version of Cas9 (dCas9) was engineered, resulting in a DNA binding protein capable of site-specific target recognition but unable to cut the DNA. By means of dCas9 fusion to heterologous domains, including transcriptional activators or repressors, specific modulation of gene expression has successfully been achieved invitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used invitro, and to our knowledge, there are no reports regarding its use for the activation of endogenous gene expression in mammalian embryos. As a proof of principle, we evaluated the CRISPR-on system in bovine embryos for modulation of endogenous expression of SMARCA4 and TFAP2C, transcription factors implicated in trophoblast lineage commitment. We hypothesised that CRISPR-on may induce SMARCA4 or TFAP2C endogenous expression, enabling the design of strategies to induce trophectoderm proliferation of invitro-derived embryos. To this aim, we designed and synthesised 4 non-overlapping single guide RNAs to target the regulatory region of each of these target genes. Presumptive zygotes were cytoplasmically microinjected with a mix containing dCas9-VP160 mRNA and a pool of 4 single guide RNAs targeting SMARCA4 (dCas9_SM group) or TFAP2C (dCas9_TF group). As control, a non-injected group was also included. Analysis was carried out in pools of 10 early embryos or 5 blastocysts and at least 3 biological replicates were included. Gene expression was assessed by RTqPCR at Days 2, 4, and 7 after microinjection and data were normalized to that obtained for the non-injected group. The CRISPR-on system was efficient to induce expression of SMARCA4 two days after microinjection (dCas9_SM group, Mann-Whitney t-test; P<0.05), but failed to significantly increase TFAP2C expression (dCas9_TF group). Surprisingly, CDX2, which is a downstream effector for trophectoderm maintenance, was induced both in dCas9_SM and dCas9_TF groups, supporting the CRISPR-mediated induction of targeted transcription factors. However, no changes were observed in the endogenous level of NANOG. Additional analysis is currently ongoing to determine whether CRISPR-on mediated induction of SMARCA4 and/or TFAP2C expression affects lineage specification and regulation. To our knowledge, this is the first report on the use of CRISPR-on for modulation of endogenous gene expression in mammalian embryos. Our study lays the foundations for CRISPR-on application in embryos as a useful tool to understand key cell fate decisions and will enable unprecedented studies of significance to embryo development, cell differentiation, and segregation.

scholarly journals AtNSE1 and AtNSE3 are required for embryo pattern formation and maintenance of cell viability during Arabidopsis embryogenesis

2019 ◽  
Vol 70 (21) ◽  
pp. 6229-6244
Author(s):  
Gang Li ◽  
Wenxuan Zou ◽  
Liufang Jian ◽  
Jie Qian ◽  
Jie Zhao
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Fate ◽  
Embryo Development ◽  
Higher Plants ◽  
Early Embryo ◽  
Embryo Cell ◽  
Early Embryogenesis ◽  
Embryo Abortion ◽  
Cell Fate Decisions

Abstract Embryogenesis is an essential process during seed development in higher plants. It has previously been shown that mutation of the Arabidopsis non-SMC element genes AtNSE1 or AtNSE3 leads to early embryo abortion, and their proteins can interact with each other directly. However, the crucial regions of these proteins in this interaction and how the proteins are cytologically involved in Arabidopsis embryo development are unknown. In this study, we found that the C-terminal including the Ring-like motif of AtNSE1 can interact with the N-terminal of AtNSE3, and only the Ring-like motif is essential for binding with three α motifs of AtNSE2 (homologous to AtMMS21). Using genetic assays and by analysing molecular markers of cell fate decisions (STM, WOX5, and WOX8) in mutant nse1 and nse3 embryos, we found that AtNSE1 and AtNSE3 work non-redundantly in early embryo development, and that differentiation of the apical meristem and the hypophysis fails in the mutants, which have disrupted auxin transportation and responses. However, the upper cells of the suspensor in the mutants seem to have proper embryo cell identity. Cytological examination showed that cell death occurred from the early embryo stage, and that vacuolar programmed cell death and necrosis in the nse1 and nse3 mutant embryos led to ovule abortion. Thus, AtNSE1 and AtNSE3 are essential for maintaining cell viability and growth during early embryogenesis. Our results improve our understanding of the functions of SMC5/6 complex in early embryogenesis in Arabidopsis.

scholarly journals Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Andrea Jurisicova ◽  
Beth M Acton
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Fate ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Culture Conditions ◽  
Early Embryo ◽  
Preimplantation Embryo Development

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

56 Nobiletin affects gene expression profiles of the ERK1/2 pathway in bovine embryos produced invitro

2021 ◽  
Vol 33 (2) ◽  
pp. 135
Author(s):  
Y. N. Cajas ◽  
K. E. Cañón-Beltrán ◽  
C. L. V. Leal ◽  
A. Gutierrez-Adán ◽  
E. González ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Cell Cycle ◽  
Embryo Development ◽  
Expression Profiles ◽  
Calf Serum ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
And Oxidative Stress

During embryo development the embryonic genome activation (EGA) is one of the most important events and in bovine embryos it occurs at the 8- to 16-cell stage. Invitro embryo production increases the levels of reactive oxygen species (ROS), which leads to the low quality of the produced blastocysts, possibly by affecting EGA. Nobiletin is an antioxidant that affects cell cycle regulation (Huang et al. 2016 Evid. Based. Complement. Alternat. Med. 2016, 2918796, https://doi.org/10.1155/2016/2918796). Therefore, we aimed to evaluate the effect of nobiletin supplementation, in two key periods of early embryo development, on blastocyst yield and expression of selected genes of the ERK1/2 pathway and oxidative stress on produced embryos. Invitro zygotes were cultured in synthetic oviductal fluid (SOF) with 5% fetal calf serum (control, C); C with 5 or 10µM nobiletin (MedChemExpress) (N5, N10); or C with 0.03% dimethyl sulfoxide (CDMSO; vehicle for nobiletin dilution) during the minor (21–54h post-insemination (hpi): 2- to 8-cell; MNEGA; 12 replicates) or major (54–96 hpi: 8- to 16-cell; MJEGA; 10 replicates) phase of EGA. The speed of development was considered and embryos that reached ≥8 cells at 54 hpi from MNEGA phase and ≥16 cells at 96 hpi from MJEGA phase, were selected and further cultured in control medium until Day 7. Embryos at ≥8 cell (MNEGA), ≥16 cell (MJEGA) stage, and Day 7 blastocysts from both periods were snap-frozen in liquid N2 for gene expression analysis (3 pools of 10 embryos/treatment). The expression of genes related to ERK1/2 pathway (H3–3B, H3–3A, NFE2L2) and oxidative stress (GPX1) were measured by quantitative PCR; H2AFZ and ACTB were used as housekeeping genes. Statistical analysis was assessed by one-way ANOVA. At 54 hpi, irrespective of nobiletin supplementation, no differences were found in the proportion of embryos that reached the 8-cell stage between groups in both phases (≈60%). At 96 hpi, nobiletin during MJEGA showed a higher proportion of embryos reaching the 16-cell stage than control groups (≈70% vs. ≈60%, respectively; P<0.001). Blastocyst yield for MNEGA and MJEGA was higher (P<0.001) for N5 (40.0±0.8% and 46.7±0.8%) and N10 (41.0±0.9% and 54.5±1.1%) compared with C (32.0±0.6% and 38.4±1.1%) and CDMSO (31.2±0.4% and 35.8±1.0%) groups, while N10 was higher (P<0.05) compared to N5 group in MJEGA. The expression of H3–3B and H3–3A were higher (P<0.05) in 8-cell embryos from N5 and N10 groups during MNEGA; while in 16-cell embryos, H3–3B and NFE2L2 were higher (P<0.05) only in the N10 group compared with both controls during MJEGA. GPX1 was upregulated in nobiletin-supplemented groups from both phases (8- and 16-cell embryos and blastocysts) compared with controls (P<0.05). In conclusion, nobiletin supplementation during minor or major EGA has a positive effect in pre-implantation embryo development and modifies the transcription of cell cycle and oxidative stress genes in early embryos. These benefits can be attributed to its bioactivity and indicate that it might be a tool to overcome EGA and ROS disorders in bovine invitro-produced embryos.This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador; FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.

scholarly journals Synergy between suppressor of Hairless and Notch in regulation of Enhancer of split m gamma and m delta expression.

1997 ◽  
Vol 17 (9) ◽  
pp. 5620-5628 ◽  
Author(s):  
D S Eastman ◽  
R Slee ◽  
E Skoufos ◽  
L Bangalore ◽  
S Bray ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Cell Fate ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
Notch Signaling Pathway ◽  
Cell Fate Decisions ◽  
Suppressor Of Hairless ◽  
Enhancer Of Split

The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.

scholarly journals GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mika J. Välimäki ◽  
Robert S. Leigh ◽  
Sini M. Kinnunen ◽  
Alexander R. March ◽  
Ana Hernández de Sande ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Reporter Gene ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Fate Decisions ◽  
Dual Reporter ◽  
Gene Regulatory ◽  
Reporter Gene Assays

AbstractBackgroundPharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.MethodsTranscription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.ResultsGATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.ConclusionsCollectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

scholarly journals Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst

2010 ◽  
Vol 18 (4) ◽  
pp. 675-685 ◽  
Author(s):  
Guoji Guo ◽  
Mikael Huss ◽  
Guo Qing Tong ◽  
Chaoyang Wang ◽  
Li Li Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Fate ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Cell Fate Decisions ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Genomic insights into chromatin reprogramming to totipotency in embryos

2018 ◽  
Vol 218 (1) ◽  
pp. 70-82 ◽  
Author(s):  
Sabrina Ladstätter ◽  
Kikuë Tachibana
Keyword(s):  
Cell Fate ◽  
Regulatory Networks ◽  
Chromatin Accessibility ◽  
Early Embryo ◽  
Epigenetic Reprogramming ◽  
Mammalian Embryo ◽  
Cell Fate Decisions ◽  
A Cell ◽  
Early Mouse ◽  
Chromatin Reprogramming

The early embryo is the natural prototype for the acquisition of totipotency, which is the potential of a cell to produce a whole organism. Generation of a totipotent embryo involves chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications. Understanding embryonic chromatin architecture and how this is related to the epigenome and transcriptome will provide invaluable insights into cell fate decisions. Recently emerging low-input genomic assays allow the exploration of regulatory networks in the sparsely available mammalian embryo. Thus, the field of developmental biology is transitioning from microscopy to genome-wide chromatin descriptions. Ultimately, the prototype becomes a unique model for studying fundamental principles of development, epigenetic reprogramming, and cellular plasticity. In this review, we discuss chromatin reprogramming in the early mouse embryo, focusing on DNA methylation, chromatin accessibility, and higher-order chromatin structure.

P–181 Morphine regulates BMP4 growth factor and is involved in in-vitro early embryo development and PGCs formation

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
I Muñoa ◽  
M Araolaza-Lasa ◽  
I Urizar-Arenaza ◽  
M Gianzo Citores ◽  
N Subiran Ciudad
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Environmental Factors ◽  
Embryo Development ◽  
Early Embryo ◽  
Epigenetic Changes ◽  
Morphine Treatment ◽  
Early Embryo Development

Abstract Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10–5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings: Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases Trial registration number Not applicable

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