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

Abstract

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.

Lateral Membrane Biogenesis in Human Bronchial Epithelial Cells Requires 190-kDa Ankyrin-G

“…Internal punctate localization of ZO-1 was observed in one cell. Such ZO-1 localization has been observed during the development of the tight junction both in cell culture as well as in vivo (29,30).…”

Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

“…A model proposed by Barwe et al (29) suggests that the Na/K-ATPase ␣ subunit is able to phosphorylate p85, which recruits this subunit to the plasma membrane and results in activation of phosphatidylinositol 3-kinase and the generation of inositol 1,4,5-trisphosphate, which induces the binding of annexin II to the Na/K-ATPase ␤ subunit cytoplasmic tail, activation of the Rac1 RhoGTPase, and modifications to the actin cytoskeleton. Studies have shown that loss of tight junction structure and increased paracellular permeability is often linked to the disruption of the circumferential actin ring that is localized at the apical pole of polarized epithelial cells (42)(43)(44). In MDCK cells, loss of tight junction permeability upon Na ϩ /K ϩ -ATPase inhibition also is associated with reduced stress fiber content and reduced RhoA activity (34).…”

The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

“…The transepithelial electrical resistance (TER) can be used to monitor the tightness of the seal between neighboring cells. Because the recruitment of Rab13 to the apicolateral junctional complex occurred at an early stage in the assembly of TJ (Zahraoui et al, 1994;Sheth et al, 2000), we postulated that it might be involved in TJ sealing. Therefore, we measured TER over a 120-h time period of parental MDCK, GFP, GFP-Rab13WT, GFP-Rab13T22N, and GFP-Rab13Q67L cells grown on filters (Figure 2).…”

“…Although the exact role of Rab proteins is still unclear, they may control the assembly of protein complexes involved in various cellular processes including vesicle movement and fusion or actin organization (Stenmark et al, 1995;Kato et al, 1996;Peränen et al, 1996;Ren et al, 1996;Echard et al, 1998;Nielsen et al, 1999;White et al, 1999). Recently, we have found that Rab13 is recruited to cell-cell contacts at an early stage of assembly of junctional complexes (Sheth et al, 2000), suggesting that Rab13 may be a good candidate for regulating the assembly cell-cell junctions. In this study, we investigate the role of Rab13 in TJ functions.…”