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

We have investigated the mechanisms by which the epithelial apicolateral ju nctional 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 int ercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membra ne contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compac tion at the 8-cell stage in which the TJ-associated peripheral membrane pro tein, ZO-1 alpha isoform, was co-localized with both alpha- and beta -caten in. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1 alpha- and other TJ proteins (ZO-1 alpha+ isoform, occludin, cingulin) colocalized in an apical TJ which was separate from a subjacent E-cadherin/catenin zon ula adherens complex. Thin-section electron microscopy confirmed that a sin gle zonula adherens-like junctional complex present at the AJC site followi ng compaction matured into a dual TJ and zonula adherens complex at the bla stocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed th at a functional TJ seal was established coincident with blastocoel formatio n. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates fr om the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1 alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm dif ferentiation 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. (C) 2000 Elsevier Science Irel and Ltd. All rights reserved.