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 protein cingulin is expressed by maternal and embryonic genomes during early mouse development

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1145-1151 ◽  
Author(s):  
Q. Javed ◽  
T.P. Fleming ◽  
M. Hay ◽  
S. Citi
Keyword(s):  
Tight Junction ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Contact ◽  
Mouse Development ◽  
Early Cleavage ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Formation

The expression of the tight junction peripheral membrane protein, cingulin (140 × 10(3) M(r), was investigated in mouse eggs and staged preimplantation embryos by immunoblotting and immunoprecipitation. Polyclonal antibody to chicken brush cingulin detected a single 140 × 10(3) M(r) protein in immunoblots of unfertilised eggs and all preimplantation stages. Relative protein levels were high in eggs and early cleavage stages, declined during later cleavage and increased again in expanding blastocysts. Quantitative immunoprecipitation of metabolically labelled eggs and staged embryos also revealed a biphasic pattern for cingulin synthesis with relative net levels being high in unfertilised eggs, minimal during early cleavage, rising 2.3-fold specifically at the onset of compaction (8-cell stage, when tight junction formation begins), and increasing further at a linear rate during morula and blastocyst stages. Cingulin synthesis in eggs is not influenced by fertilisation (or aging, if unfertilised), but this level declines sharply after first cleavage. These results indicate that cingulin is expressed by both maternal and embryonic genomes. The turnover of maternal cingulin (unfertilised eggs) and embryonic cingulin at a stage before tight junction formation begins (4-cell stage) is higher (t1/2 approximately 4 hours) than cingulin synthesised after tight junction formation (blastocysts; t1/2 approximately 10 hours). This increase in cingulin stability is reversed in the absence of extracellular calcium. Cingulin synthesis is also tissue-specific in blastocysts, being up-regulated in trophectoderm and down-regulated in the inner cell mass. Taken together, the results suggest that (i) cingulin may have a role during oogenesis and (ii) cell-cell contact patterns regulate cingulin biosynthesis during early morphogenesis, contributing to lineage-specific epithelial maturation.

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 Transitions in cell potency during early mouse development are driven by Notch

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergio Menchero ◽  
Isabel Rollan ◽  
Antonio Lopez-Izquierdo ◽  
Maria Jose Andreu ◽  
Julio Sainz de Aja ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Types ◽  
Mammalian Development ◽  
Mouse Development ◽  
Cell Stage ◽  
Notch Signalling Pathway ◽  
Gradual Loss ◽  
Early Mouse

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

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.

scholarly journals Transitions in cell potency during early mouse development are driven by Notch

2018 ◽  
Author(s):  
Sergio Menchero ◽  
Antonio Lopez-Izquierdo ◽  
Isabel Rollan ◽  
Julio Sainz de Aja ◽  
Maria Jose Andreu ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Types ◽  
Mammalian Development ◽  
Mouse Development ◽  
Cell Stage ◽  
Notch Signalling Pathway ◽  
Gradual Loss ◽  
Early Mouse

AbstractThe Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

Cell division and cell allocation in early mouse development

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 37-51
Author(s):  
S. J. Kelly ◽  
J. G. Mulnard ◽  
C. F. Graham
Keyword(s):  
Cell Division ◽  
Tritiated Thymidine ◽  
Mouse Embryos ◽  
Mouse Development ◽  
Stages Of Development ◽  
Cell Stage ◽  
Cell Cycles ◽  
Short Cell ◽  
Early Mouse

Cell division was observed in intact and dissociated mouse embryos between the 2-cell stage and the blastocyst in embryos developing in culture. Division to the 4-cell stage was usually asynchronous. The first cell to divide to the 4-cell stage produced descendants which tended to divide ahead of those cells produced by its slow partner at all subsequent stages of development up to the blastocyte stage. The descendants of the first cell to divide to the 4-cell stage did not subsequently have short cell cycles. The first cell or last cell to divide from the 4-cell stage was labelled with tritiated thymidine. The embryo was reassembled, and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell stage.

A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1353-1361
Author(s):  
J.M. Baltz ◽  
J.D. Biggers ◽  
C. Lechene
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
K Concentration ◽  
Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

scholarly journals Tight and Adherens Junctions in the Ovine Uterus: Differential Regulation by Pregnancy and Progesterone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3922-3931 ◽  
Author(s):  
M. Carey Satterfield ◽  
Kathrin A. Dunlap ◽  
Kanako Hayashi ◽  
Robert C. Burghardt ◽  
Thomas E. Spencer ◽  
...  
Keyword(s):  
Tight Junction ◽  
Adherens Junctions ◽  
Luminal Epithelium ◽  
Tissue Fluid ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Conceptus Development ◽  
Associated Proteins ◽  
Continuous Presence ◽  
D 12

In species with noninvasive implantation by conceptus trophectoderm, fetal/maternal communications occur across the endometrial epithelia. The present studies identified changes in junctional complexes in the ovine endometrium that regulate paracellular trafficking of water, ions, and other molecules, and the secretory capacity of the uterine epithelia. Distinct temporal and spatial alterations in occludin, tight junction protein 2, and claudin 1–4 proteins were observed in the endometrium of cyclic and early pregnant ewes. Dynamic changes in tight junction formation were characterized by an abundance of tight junction proteins on d 10 of the estrous cycle and pregnancy that substantially decreased by d 12. Early progesterone administration advanced conceptus development on d 9 and 12 that was associated with loss of tight-junction-associated proteins. Pregnancy increased tight-junction-associated proteins between d 14–16. Cadherin 1 and β-catenin, which form adherens junctions, were abundant in the endometrial glands, but decreased after d 10 of pregnancy in the luminal epithelium and then increased by d 16 with the onset of implantation. Results support the ideas that progesterone elicits transient decreases in tight and adherens junctions in the endometrial luminal epithelium between d 10–12 that increases selective serum and tissue fluid transudation to enhance blastocyst elongation, which is subsequently followed by an increase in tight and adherens junctions between d 14–16 that may be required for attachment and adherence of the trophectoderm for implantation. The continuous presence of tight and adherens junctions in the uterine glands would allow for vectorial secretion of trophic substances required for conceptus elongation and survival.

Focal adhesion kinase PTK2 autophosphorylation is not required for the activation of sodium–hydrogen exchange by decreased cell volume in the preimplantation mouse embryo

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 173-179
Author(s):  
Jane C. Fenelon ◽  
Baozeng Xu ◽  
Jay M. Baltz
Keyword(s):  
Focal Adhesion Kinase ◽  
Cell Volume ◽  
Focal Adhesion ◽  
Mouse Embryo ◽  
Hydrogen Exchange ◽  
Cell Types ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Early Embryos ◽  
Early Mouse

SummaryRecovery from decreased cell volume is accomplished by a regulated increase of intracellular osmolarity. The acute response is activation of inorganic ion transport into the cell, the main effector of which is the Na+/H+ exchanger NHE1. NHE1 is rapidly activated by a cell volume decrease in early embryos, but how this occurs is incompletely understood. Elucidating cell volume-regulatory mechanisms in early embryos is important, as it has been shown that their dysregulation results in preimplantation developmental arrest. The kinase JAK2 has a role in volume-mediated NHE1 activation in at least some cells, including 2-cell stage mouse embryos. However, while 2-cell embryos show partial inhibition of NHE1 when JAK2 activity is blocked, NHE1 activation in 1-cell embryos is JAK2-independent, implying a requirement for additional signalling mechanisms. As focal adhesion kinase (FAK aka PTK2) becomes phosphorylated and activated in some cell types in response to decreased cell volume, we sought to determine whether it was involved in NHE1 activation in the early mouse embryo. FAK activity requires initial autophosphorylation of a tyrosine residue, Y397. However, FAK Y397 phosphorylation levels were not increased in either 1- or 2-cell embryos after cell volume was decreased. Furthermore, the selective FAK inhibitor PF-562271 did not affect NHE1 activation at concentrations that essentially eliminated Y397 phosphorylation. Thus, autophosphorylation of FAK Y397 does not appear to be required for NHE1 activation induced by a decrease in cell volume in early mouse embryos.

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