TIGHT JUNCTION ASSEMBLY DURING MOUSE BLASTOCYST FORMATION IS REGULATED BY LATE EXPRESSION OF ZO-1 ALPHA(+) ISOFORM

The mouse preimplantation embryo has been used to investigate the de n ovo synthesis of tight junctions during trophectoderm epithelial diffe rentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembl y of the cytoplasmic plaque protein ZO-1 occurring 12 hours before tha t of cingulin, Subsequently, two alternatively spliced isoforms of ZO- 1 (alpha(+) and alpha(-)), differing in the presence or absence of an 80 residue a domain were reported. Here, the temporal and spatial expr ession of these ZO-1 isoforms has been investigated at different stage s of preimplantation development, ZO-1 alpha(-) mRNA was present in oo cytes and all preimplantation stages, whilst ZO-1 alpha(+) transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in tr ophectoderm and ICM cells, Immunoprecipitation of S-35-labelled embryo s also showed synthesis of ZO-1 alpha(-) throughout cleavage, whereas synthesis of ZO-1 alpha(+) was only apparent from the blastocyst stage , In addition, P-33-labelling showed both isoforms to be phosphorylate d at the early blastocyst stage, The pattern and timing of membrane as sembly of the two isoforms was also distinct, ZO-1 alpha(-) was initia lly seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the membrane unti l they completely surrounded each cell with a zonular belt by the late morula stage, ZO-1 alpha(+) however, was first seen as perinuclear fo ci in late morulae before assembling at the tight junction, Membrane a ssembly of ZO-1 alpha(+) first occurred during the 32-cell stage and w as zonular just prior to the early blastocyst stage. Immuno-staining i ndicative of both isoforms was restricted to the trophectoderm lineage , Membrane assembly of ZO-1 alpha(+) and blastocoele formation were se nsitive to brefeldin A, an inhibitor of intracellular trafficking beyo nd the Golgi complex, In addition, the tight junction transmembrane pr otein occludin co-localised with ZO-1 alpha(+) at the perinuclear site s in late morulae and at the newly assembled cell junctions. These res ults provide direct evidence from a native epithelium that ZO-1 isofor ms perform distinct roles in tight junction assembly. Moreover, the la te expression of ZO-1 alpha(+) and its apparent intracellular interact ion with occludin may act as a final rate-limiting step in the synthes is of the tight junction, thereby regulating the time of junction seal ing and blastocoele formation in the early embryo.