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.

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.

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 PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 874-885 ◽  
Author(s):  
Qian Wang ◽  
Xiao-Wei Chen ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Darby Canine Kidney ◽  
E Cadherin

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

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 Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1355-1367 ◽  
Author(s):  
P.A. De Sousa ◽  
G. Valdimarsson ◽  
B.J. Nicholson ◽  
G.M. Kidder
Keyword(s):  
Gap Junction ◽  
Plasma Membranes ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Brefeldin A ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Cell Fractionation ◽  
Cell Stage ◽  
Junction Protein

Gap junction assembly in the preimplantation mouse embryo is a temporally regulated event, beginning a few hours after the third cleavage during the morphogenetic event known as compaction. Recently, we demonstrated that both mRNA and protein corresponding to connexin43, a gap junction protein, accumulate through preimplantation development beginning at least as early as the 4-cell stage. Using an antibody raised against a synthetic C-terminal peptide of connexin43, this protein was shown to assemble into gap junction-like plaques beginning at compaction (G. Valdimarsson, P. A. De Sousa, E. C. Beyer, D. L. Paul and G. M. Kidder (1991). Molec. Reprod. Dev. 30, 18–26). The purpose of the present study was to follow the fate of nascent connexin43 during preimplantation development, from synthesis to plaque insertion, and to learn more about the control of gap junction assembly during compaction. Cell fractionation and reverse transcription-polymerase chain reaction were employed to show that connexin43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of connexin43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent connexin43 was then followed throughout preimplantation development by means of laser confocal microscopy, using two other peptide (C-terminal)-specific antibodies. As was reported previously, connexin43 could first be detected in gap junction-like plaques beginning in the 8-cell stage, at which time considerable intracellular immunoreactivity could be seen as well. Later, connexin43 becomes differentially distributed in the apposed plasma membranes of morulae and blastocysts: a zonular distribution predominates between outside blastomeres and trophectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic immunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with cycloheximide, and redistributed by treatment with monensin or brefeldin-A, known inhibitors of protein trafficking. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the regulated step in the onset of gap junction assembly during compaction is downstream of transcription and translation and involves mobilization of connexin43 through trafficking organelles to plasma membranes.

scholarly journals 236.Differential expression of monocarboxylate cotransporter proteins in preimplantation embryos

2004 ◽  
Vol 16 (9) ◽  
pp. 236 ◽  
Author(s):  
S. Jansen ◽  
M. Pantaleon ◽  
P. Kaye
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Confocal Laser ◽  
Cell Contact ◽  
Cleavage Stage ◽  
Mouse Development

During preimplantation development mouse embryos demonstrate a switch in substrate preference. Pyruvate consumption, high during the first few cleavage stages, declines as the morula develops to a blastocyst, when glucose becomes the preferred substrate. Whilst pyruvate utilisation has been well characterised, changes in the function and expression of pyruvate transporters during this crucial period remain unclear. Pyruvate, lactate and other monocarboxylates are transported across mammalian cell membranes via a specific H+-monocarboxylate cotransporter (MCT). Fourteen members of this family have been identified of which MCT1, MCT2 and MCT4 are well characterised. Although mRNA expression profiles are known during early mouse development (1,2), the specific roles of each protein isoform are unknown. In order to understand these, the expression pattern for each isoform and their cellular localisation during preimplantation development have been determined. Mouse embryos were freshly collected from superovulated Quackenbush mice at 24, 48, 72 and 96 h post-hCG and expression of MCT1, MCT2 and MCT4 analysed by confocal laser scanning immunohistochemistry. Our results confirm that all three MCT proteins are expressed in preimplantation embryos. Immunoreactivity for MCT1 and MCT2 appears diffuse throughout the cytoplasm of cleavage stage embryos. As development proceeds, MCT1 localised to the basolateral membranes of morulae and blastocysts, whilst stronger MCT2 expression was found on the apical trophectoderm as well as the inner cell mass. MCT4 immunoreactivity on the other hand is apparent at cell-cell contact sites in cleavage stage embryos and morulae, but it is not apparent in the blastocyst. The demonstration of different expression patterns for MCT1, MCT2 and MCT4 in mouse embryos implies specific functional roles for each in the critical regulation of H+, pyruvate and lactate transport during preimplantation development. (1) Harding EA, Day ML, Gibb CA, Johnson MH, Cook DI (1999) The activity of the H+-monocarboxylate cotransporter during pre-implantation development in the mouse. Eur. J. Physiol. 438, 397–404. (2) H�rubel F, El Mouatassim S, Gu�rin P, Frydman R, M�n�zo Y (2002) Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development. Zygote 10, 175–181.

scholarly journals PATJ regulates tight junction formation and polarity in mammalian epithelial cells

2005 ◽  
Vol 168 (5) ◽  
pp. 705-711 ◽  
Author(s):  
Kunyoo Shin ◽  
Sam Straight ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Complex Function ◽  
Cell Polarization ◽  
Tight Junction Protein ◽  
Apical Region ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Formation

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs–PALS1–PATJ complex function in epithelial polarity.

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