Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.     

Assembly of tight junctions during early vertebrate development

Tight junction formation during development is critical for embryonic patterning and organization. We consider mechanisms of junction biogenesis in cleaving mouse and Xenopus eggs. Junction assembly follows the establishment of cell polarity at 8-cell (mouse) or 2-cell (Xenopus) stages, characterized by sequential membrane delivery of constituents, coordinated by embryonic (mouse) or maternal (Xenopus) expression programmes. Cadherin adhesion is permissive for tight junction construction only in the mouse. Occludin post-translational modification and membrane delivery, mediated by delayed ZO-1 alpha(+)isoform expression in the mouse, provides a mechanism for completion of tight junction biogenesis and sealing, regulating the timing of blastocoel cavitation.     

Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation

The mouse blastocyst forms during the 32-cell stage with the emergence of the blastocoelic cavity. This developmental transition is dependent upon the differentiation and transport function of the trophectoderm epithelium which forms the wall of the blastocyst and exhibits functional intercellular tight junctions (TJs) to maintain epithelial integrity during blastocoele expansion. To investigate mechanisms regulating the timing of blastocyst formation, we have examined the dynamics of expression of occludin, an integral membrane protein of the TJ. Confocal microscopy of intact embryos and synchronised cell clusters revealed that occludin first assembles at the apicolateral membrane contact site between nascent trophectoderm cells usually during the early 32-cell stage, just prior to the time of blastocoele cavitation. This is a late event in the assembly of TJ-associated proteins within trophectoderm which, from our previous data, spans from 8- to 32-cell stages. Occludin membrane assembly is dependent upon prior E-cadherin-mediated cell-cell adhesion and is sensitive to brefeldin A, an inhibitor of Golgi-to-membrane transport. Occludin is delivered to the TJ site in association with the TJ plaque protein, ZO-1(&agr;)+, which we have shown previously is newly transcribed and translated during late cleavage. Immediately after assembly and before cavitation, occludin localised at the TJ site switches from a Triton X-100-soluble to -insoluble form indicative of actin cytoskeletal and/or membrane anchorage. Occludin mRNA and protein are detectable throughout cleavage by RT-PCR and immunoblotting, respectively, indicating that timing of membrane assembly is not controlled by expression alone. Rather, we have identified changes in the pattern of different occludin forms expressed during cleavage which, using phosphatase treatment of embryo lysates, include post-translational modifications. We propose that the phosphorylation of one form of occludin (band 2, 65-67 kDa) during late cleavage, which leads to its exclusive conversion from a Triton X-100-soluble to -insoluble pool, may regulate occludin association with ZO-1(&agr;)+ and membrane assembly, and thereby act to control completion of TJ biogenesis and the timing of blastocyst formation.     

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.     

Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo

In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.     

Dynamic assembly of tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin during mouse tooth development

Tight junctions might play a role during tissue morphogenesis and cell differentiation. In order to address these questions, we have studied the distribution pattern of the tight junction-associated proteins ZO-1, ZO-2, ZO-3 and occludin in the developing mouse tooth as a model. A specific temporal and spatial distribution of tight junction-associated proteins during tooth development was observed. ZO-1 appeared discontinuously in the cell membrane of enamel organ and dental mesenchyme cells. However, endothelial cells of the dental mesenchyme capillaries displayed a continuous fluorescence at the cell membrane. Inner dental epithelium first showed an evident signal for ZO-1 at the basal pole of the cells at bud/cap stage, but ZO-1 was accumulated at the basal and apical pole of preameloblast/ameloblasts at late bell stage. Surprisingly, in the incisor ZO-1 decreased as the inner dental epithelium differentiated, and was re-expressed in secretory and mature ameloblasts. On the contrary, ZO-2 was confined to continuous cell-cell contacts of the enamel organ in both molars and incisors. The lateral cell membrane of inner dental epithelial cells was specifically ZO-2 labeled. However, ZO-3 was expressed in oral epithelium whereas dental embryo tissues were negative. In addition, occludin was hardly detected in dental tissues at the early stage of tooth development, but was distributed continuously at the cell membrane of endothelial cells of ED19.5 dental mesenchyme. In incisors, occludin was detected at the cell membrane of the secretory pole of ameloblasts. The occurrence and relation during tooth development of tight junction proteins ZO-1, ZO-2 and occludin, but not ZO-3, suggests a combinatory assembly in tooth morphogenesis and cell differentiation.     

Molecular maturation of cell adhesion systems during mouse early development

During cleavage, the mouse embryo expresses a variety of cell adhesion systems on its cell surfaces. We have reviewed biogenetic and assembly criteria for the formation of the uvomorulin/catenin, tight junction and desmosome adhesion systems as the trophectoderm differentiates. Each system reveals different mechanisms regulating molecular maturation. Adhesion processes contribute to the generation of distinct tissues in the blastocyst by modifying the expression pattern of blastomeres entering the non-epithelial inner cell mass lineage. Cell adhesion also influences the spatial organisation, but rarely the timing of expression, of proteins involved in trophectoderm differentiation.     

Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin

Compaction of the eight-cell stage mouse embryo is a critical event in the generation of different cell types within the preimplantation embryo. Uvomorulin, a member of the cadherin family of cell adhesion molecules, is important during compaction and its phosphorylation increases early in the eight-cell stage, suggesting that this posttranslational modification may be important for compaction to proceed. We have assessed the importance of the phosphorylation of uvomorulin during compaction by preventing, reversing, or inducing adhesion prematurely. The only condition that affected the overall level of uvomorulin phosphorylation was the prevention of compaction through prolonged exposure of four-cell embryos to low Ca²⁻. This treatment reduced the level of uvomorulin phosphorylation in eight-cell embryos, and perturbed its localization to regions of cell-cell contact. Thus, whilst the phosphorylation of uvomorulin does not appear to regulate directly uvomorulin's adhesive function, it may be associated with the redistribution of uvomorulin during compaction. © 1996 Wiley-Liss, Inc.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Tight junction biogenesis in the early Xenopus embryo

Tight junctions (TJs) perform a critical role in the transport functions and morphogenetic activity of the primary epithelium formed during Xenopus cleavage. Biogenesis of these junctions was studied by immunolocalization of TJ-associated proteins (cingulin, ZO-1 and occludin) and by an in vivo biotin diffusion assay. Using fertilized eggs synchronized during the first division cycle, we found that membrane assembly of the TJ initiated at the animal pole towards the end of zygote cytokinesis and involved sequential incorporation of components in the order cingulin, ZO-1 and occludin. The three constituents appeared to be recruited from maternal stores and were targeted to the nascent TJ site by different pathways. TJ protein assembly was focused precisely to the border between the oolemma-derived apical membrane and newly-inserted basolateral membrane generated during cytokinesis and culminated in the formation of functional TJs in the two-cell embryo, which maintained a diffusion barrier. New membrane formation and the generation of cell surface polarity therefore precede initiation of TJ formation. Moreover, assembly of TJ marker protein precisely at the apical-basolateral membrane boundary was preserved in the complete absence of intercellular contacts and adhesion. Thus, the mechanism of TJ biogenesis in the Xenopus early embryo relies on intrinsic cues of a cell autonomous mechanism. These data reveal a distinction between Xenopus and mammalian early embryos in the origin and mechanisms of epithelial cell polarization and TJ formation during cleavage of the egg.     

Cell allocation in half- and quadruple-sized preimplantation mouse embryos

The size of preimplantation mouse embryos was experimentally manipulated in order to examine the consequences for the allocation of cells to the two primary tissues, trophectoderm and inner cell mass (ICM). Half embryos were produced by the mechanical lysis of one cell at the two-cell stage and quadruple embryos by the aggregation of four whole eight-cell embryos. Such procedures are shown not only to alter the absolute number of cells that are assigned to the trophectoderm and ICM, but also to disturb significantly the proportions of these two tissues in the blastocyst. The proportion of trophectoderm is directly related to the surface area of the morula, as is predicted by a purely epigenetic scheme for cell allocation.     

Spatial patterns of gene expression in preimplantation mouse embryos

The distribution of total polyadenylated RNA and mRNAs from the beta-actin, fibronectin, and cytokeratin Endo A genes was examined in preimplantation mouse embryos using in situ hybridization of riboprobes to RNA in sections of embryos. Polyadenylated RNA was found in the cytoplasm of all cells of blastocyst-stage embryos, whereas the specific mRNAs displayed three distinct patterns of expression: uniform throughout the embryo (beta-actin), enriched in the inner cell mass (fibronectin), and enriched in the trophectoderm (Endo A). In eight-cell embryos, the polyadenylated RNA was more concentrated in nuclei than in the cytoplasm (as noted previously), although this was not the case in blastocysts, nor was it true for the specific mRNAs that were examined. These experiments demonstrate that there is localized gene expression in the early mouse embryo, which correlates with the formation of the trophectoderm and the inner cell mass.     

Functions of the IGFs in early mammalian development

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein databases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action. © 1993 Wiley-Liss, Inc.     

Disruption of the cytokeratin filament network in the preimplantation mouse embryo

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Qualitative patterns of protein synthesis in the preimplantation mouse embryo: I. Normal pregnancy

Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged.Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of l-[³⁵S]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation.