B. Sheth et al., TIGHT JUNCTION ASSEMBLY DURING MOUSE BLASTOCYST FORMATION IS REGULATED BY LATE EXPRESSION OF ZO-1 ALPHA(+) ISOFORM, Development, 124(10), 1997, pp. 2027-2037
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.