Recent studies in cultured epithelial cells have indicated the significance of ZO proteins in epithelial morphogenesis and junctional biology, in particular ZO-1 and ZO-2. The dual suppression by gene-deletion and protein-depletion of ZO-1 and ZO-2, respectively, in mouse mammary epithelial cell-line EpH4 was sufficient to abolish the assembly of TJ strands and thus, the permeability barrier function. In this context, exogenous expression of either two proteins rescued the mutant phenotype,Anemarsaponin-E thus exhibiting functional redundancy of ZO-1 and ZO-2. Notably, the absence of TJs did not affect apico-basal polarization. In a similar study done on canine kidney epithelial cell-line MDCK, although TJ presence was not abolished, protein-depletion of both ZO-1 and ZO-2 led to increased macromolecular solute permeability and abnormal barrier remodeling kinetics. In addition, the organization of the apical circumferential actomyosin ring was compromised. This was associated with an irregular epithelium organization in which cells were laterally misaligned and the apical domain was distended. The importance of these two ZO proteins is further emphasized in in vivo mouse models in which either ZO-1 or ZO-2 gene knockouts resulted in embryonic lethality. ZO-1 gene-deleted mice were embryonic lethal at E10.5 to E11.5. This was associated with a defective organization of the notochord,Anemarsaponin-BIII neural tube and allantois, resulting in extensive apoptosis. Additionally, angiogenesis in the yolk sac was deficient despite normal endothelial cell layer differentiation. Similarly, ZO-2 null mutant mice also exhibited embryonic lethality, but at an earlier stage of developmental arrest from E6.5 compared with ZO-1 null mice. This earlier lethality is possibly due to a compromised TJ structure and barrier function, which in contrast remained unaltered in the ZO1 null. ZO-2 null lethality was preceded by a reduced proliferation of the embryo at early implantation E6.5 and increased apoptosis at E7.5, leading in failure to gastrulate. Contrary to both ZO1 and ZO-2 null phenotypes, ZO-3 gene-deletion in mouse and cell-line models revealed no observable abnormalities, indicating a dispensable function of ZO-3.