scholarly journals Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein, ZO-1.

1989 ◽  
Vol 108 (4) ◽  
pp. 1407-1418 ◽  
Author(s):  
T P Fleming ◽  
J McConnell ◽  
M H Johnson ◽  
B R Stevenson
Keyword(s):  
Cell Adhesion ◽  
Tight Junction ◽  
De Novo ◽  
Specific Protein ◽  
Early Embryo ◽  
Microtubule Depolymerization ◽  
Cell Stage ◽  
Surface Distribution ◽  
Dna And Rna ◽  
Mouse Early Embryo

Tight junction development during trophectoderm biogenesis in the mouse preimplantation embryo has been examined using monoclonal antibodies recognizing the tight junction-specific peripheral membrane protein, ZO-1. In immunoblots, mouse embryo ZO-1 had a molecular mass (225 kD) equivalent to that in mouse liver, was barely detectable in four-cell embryos although later stages exhibited increasing levels. ZO-1 was first detected immunocytochemically at the compacting eight-cell stage, coincident with or just after the expression of basolateral cell adhesion and apical microvillous polarity. Initially, ZO-1 was present as a series of spots along the boundary between free and apposed cell surfaces in intact embryos or cell couplets, but subsequently staining became more linear with blastocyst trophectoderm cells being bordered by a continuous ZO-1 belt. Inhibition of cell adhesion at the 8-cell stage delayed ZO-1 appearance and randomized its surface distribution in a reversible manner. Microfilament disruption, but not microtubule depolymerization, produced major disturbances in ZO-1 distribution. ZO-1 assembly de novo appeared to be independent of proximate DNA and RNA synthesis but was inhibited substantially in the absence of protein synthesis during the eight-cell stage, a treatment that did not prevent intercellular adhesion and polarization. ZO-1 surface assembly, but not adhesion and polarization, was also perturbed when single eight-cells were combined with single four-cells. The results suggest that tight junction development in mouse embryos is a secondary event in epithelial biogenesis, being dependent upon cell adhesion and cytoskeletal activity for normal expression, and can be disrupted without disturbing the generation of a stably polarized phenotype.

Localisation of tight junction protein cingulin is temporally and spatially regulated during early mouse development

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1135-1144 ◽  
Author(s):  
T.P. Fleming ◽  
M. Hay ◽  
Q. Javed ◽  
S. Citi
Keyword(s):  
Tight Junction ◽  
Cell Types ◽  
Early Embryo ◽  
Mouse Development ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Site ◽  
Mouse Early Embryo ◽  
Early Mouse

The molecular maturation of the tight junction in the mouse early embryo has been investigated by monitoring the distribution of cingulin, a 140 × 10(3) M(r) peripheral (cytoplasmic) membrane constituent of the junction, at different stages of development and in different experimental situations. Although tight junction formation does not begin until compaction at the 8-cell stage, cingulin is detectable in oocytes and all stages of cleavage, a factor consistent with our biochemical analysis of cingulin expression (Javed et al., 1992, Development 117, 1145–1151). Using synchronised egg and embryo stages and isolated cell clusters, we have identified three sites where cingulin is localised, the cytocortex, punctate cytoplasmic foci and tight junctions themselves. Cytocortical cingulin is present at the cumulus-oocyte contact site (both cell types), in unfertilised and fertilised eggs and in cleavage stages up to 16-cell morulae, particularly at microvillous domains on the embryo outer surface (eg. apical poles at compaction). Embryo manipulation experiments indicate that cortical cingulin is labile and dependent upon cell interactions and therefore is not merely an inheritance from the egg. Cingulin cytoplasmic foci are evident only in outer cells (prospective trophectoderm) from the 32-cell stage, just prior to cavitation, and decline from approx. 8 hours after cavitation has initiated. The appearance of these foci is insensitive to cycloheximide treatment and they colocalise with apically derived endocytic vesicles visualised by FITC-dextran, indicating that the foci represent the degradation of cytocortical cingulin by endocytic turnover. Cingulin is detectable at the tight junction site between blastomeres usually from the 16-cell stage, although earlier assembly occurs in a minority (up to 20%) of specimens. Cingulin assembly at the tight junction is sensitive to cycloheximide and is identifiable approx. 10 hours after cell adhesion is initiated and ZO-1 protein assembles. Collectively, our results indicate that (i) cingulin from nonjunctional sites does not contribute to tight junction assembly and (ii) the molecular maturation of the junction appears to occur progressively over at least two cell cycles.

Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 2027-2037 ◽  
Author(s):  
B. Sheth ◽  
I. Fesenko ◽  
J.E. Collins ◽  
B. Moran ◽  
A.E. Wild ◽  
...  
Keyword(s):  
Tight Junction ◽  
De Novo ◽  
Transmembrane Protein ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Cell Junctions ◽  
Cell Stage ◽  
Membrane Assembly ◽  
Morula Stage ◽  
Late Expression

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-), differing in the presence or absence of an 80 residue alpha domain were reported. Here, the temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA was present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also showed synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ was only apparent from the blastocyst stage. In addition, 33P-labelling showed both isoforms to be phosphorylated at the early blastocyst stage. The pattern and timing of membrane assembly of the two isoforms was also distinct. ZO-1alpha- was initially seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the membrane until they completely surrounded each cell with a zonular belt by the late morula stage. ZO-1alpha+ however, was first seen as perinuclear foci in late morulae before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurred during the 32-cell stage and was zonular just prior to the early blastocyst stage. Immunostaining indicative of both isoforms was restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation were sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. In addition, the tight junction transmembrane protein occludin co-localised with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. Moreover, the late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo.

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

scholarly journals Tissue-specific control of expression of the tight junction polypeptide ZO-1 in the mouse early embryo

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 295-304 ◽  
Author(s):  
T.P. Fleming ◽  
M.J. Hay
Keyword(s):  
Tight Junction ◽  
Contact Area ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Subsequent Development ◽  
Early Embryo ◽  
Cell Contact ◽  
Down Regulation ◽  
Cell Stage ◽  
Daughter Cells

The processes governing differential protein expression in preimplantation lineages were investigated using a monoclonal antibody recognising the tight junction polypeptide, ZO-1. ZO-1 localises to the maturing tight junction membrane domain in the polarised trophectoderm lineage from compaction (8-cell stage) onwards, ultimately forming a zonular belt around each trophectoderm cell of the blastocyst (32- to 64-cell stage). The protein is usually undetectable within the inner cell mass (ICM) although, in a minority of embryos, punctate ZO-1 sites are present on the surface of one or more ICM cells. Since ICM cells derive from the differentiative division of polarised 8- and 16-cell blastomeres, the distribution of ZO-1 following differentiative division in isolated, synchronised cell clusters of varying size, was examined. In contrast to the apical cytocortical pole, ZO-1 was found to be inherited by nonpolar (prospective ICM) as well as polar (prospective trophectoderm) daughter cells. Following division, polar cells adhere to and gradually envelop nonpolar cells. Prior to envelopment, ZO-1 localises to the boundary between the contact area and free membrane of daughter cells, irrespective of their phenotype. After envelopment, polar cells retain these ZO-1 contact sites whilst nonpolar cells lose them, in which case ZO-1 transiently appears as randomly-distributed punctate sites on the membrane before disappearing. Thus, symmetrical cell contact appears to initiate ZO-1 down-regulation in the ICM lineage. The biosynthetic level at which ZO-1 down-regulation occurs was investigated in immunosurgically isolated ICMs undergoing trophectoderm regeneration. By 6 h in culture, isolated ICMs generated a zonular network of ZO-1 at the contact area between outer cells, thereby demonstrating the reversibility of down-regulation. This assembly process was unaffected by alpha-amanitin treatment but was inhibited by cycloheximide. These results indicate that the ICM inherits and stabilises ZO-1 transcripts which can be utilised for rapid synthesis and assembly of the protein, a capacity that may have significance both in maintaining lineage integrity within the blastocyst and in the subsequent development of the ICM.

scholarly journals Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage

Cell Research ◽  
2002 ◽  
Vol 12 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Pan YONG ◽  
Zheng GU ◽  
Jin Ping LUO ◽  
Jun Ru WANG ◽  
Jia Ke TSO
Keyword(s):  
Embryo Development ◽  
Early Embryo ◽  
Cell Stage ◽  
Early Embryo Development ◽  
Mouse Early Embryo

scholarly journals Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Julien Richard Albert ◽  
Wan Kin Au Yeung ◽  
Keisuke Toriyama ◽  
Hisato Kobayashi ◽  
Ryutaro Hirasawa ◽  
...  
Keyword(s):  
De Novo ◽  
Cpg Islands ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mass Spectrometry Analysis ◽  
Paternal Allele ◽  
Sequencing Data ◽  
Cell Stage ◽  
Paternal Genome ◽  
Spectrometry Analysis

Abstract De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

Epithelial differentiation and intercellular junction formation in the mouse early embryo

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 105-112
Author(s):  
Tom P. Fleming ◽  
Qamar Javed ◽  
Mark Hay
Keyword(s):  
Tight Junction ◽  
Intercellular Junctions ◽  
Early Embryo ◽  
Control Mechanisms ◽  
Tight Junction Proteins ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Junction Proteins

Trophectoderm differentiation during blastocyst formation provides a model for investigating how an epithelium develops in vivo. This paper briefly reviews our current understanding of the stages of differentiation and possible control mechanisms. The maturation of structural intercellular junctions is considered in more detail. Tight junction formation, essential for blastocoele cavitation and vectorial transport activity, begins at compaction (8-cell stage) and appears complete before fluid accumulation begins a day later (approx 32-cell stage). During this period, initial focal junction sites gradually extend laterally to become zonular and acquire the peripheral tight junction proteins ZO-1 and cingulin. Our studies indicate that junction components assemble in a temporal sequence with ZO-1 assembly preceding that of cingulin, suggesting that the junction forms progressively and in the ‘membrane to cytoplasm’ direction. The protein expression characteristics of ZO-1 and cingulin support this model. In contrast to ZO-1, cingulin expression is also detectable during oogenesis where the protein is localised in the cytocortex and in adjacent cumulus cells. However, maternal cingulin is metabolically unstable and does not appear to contribute to later tight junction formation in trophectoderm. Cell-cell interactions are important regulators of the level of synthesis and state of assembly of tight junction proteins, and also control the tissue-specificity of expression. In contrast to the progressive nature of tight junction formation, nascent desmosomes (formed from cavitation) appear mature in terms of their substructure and composition. The rapidity of desmosome assembly appears to be controlled by the time of expression of their transmembrane glycoprotein constitutents; this occurs later than the expression of more cytoplasmic desmosome components and intermediate filaments which would therefore be available for assembly to occur to completion.

scholarly journals Maternal DNMT3A-dependent de novo methylation of the zygotic paternal genome inhibits gene expression in the early embryo

2020 ◽  
Author(s):  
Julien Richard Albert ◽  
Wan Kin Au Yeung ◽  
Keisuke Toriyama ◽  
Hisato Kobayashi ◽  
Ryutaro Hirasawa ◽  
...  
Keyword(s):  
De Novo ◽  
Cpg Islands ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mass Spectrometry Analysis ◽  
Paternal Allele ◽  
Cell Stage ◽  
Paternal Genome ◽  
Spectrometry Analysis ◽  
Genomic Regions

ABSTRACTDe novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. Following fertilization, the paternal genome undergoes widespread DNAme loss before the first S-phase. Paradoxically, recent mass spectrometry analysis revealed that a low level of de novo DNAme occurs exclusively on the zygotic paternal genome. However, the loci involved and impact on genic transcription was not addressed. Here, we employ allele-specific analysis of wholegenome bisulphite sequencing (WGBS) data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome in 2-cell embryos. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with zygotic paternal DNAme acquisition (PDA), many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Strikingly, PDA is lost following maternal deletion of Dnmt3a. Furthermore, a subset of promoters showing PDA which are normally transcribed from the paternal allele in blastocysts show premature transcription at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover an unexpected role for maternal DNMT3A activity in postfertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.

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