Abstract
The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.
Highlights
The epithelial tissue is one of the main types of tissue in the human body
The Zonula Occludens (ZO)-1-/- and ZO-1-/- ZO-2-/- mouse embryonic stem cells (mESC) ScaIdigests were positive for the ZO-1 mutant but not WT allele, whereas the reverse was seen with the WT and ZO-2-/- mESCs (Fig. 1B, panel a)
Since we previously reported that ZO-1 affects self-renewal and differentiation of mESCs [26], it was important to determine if the absence of ZO-1 in mESCs could impede the differentiation of embryoid bodies (EB) inner cell mass (ICM) cells into the extraembryonic endoderm (ExEn) epithelium and mistakenly lead to the interpretation of supposed ExEn abnormality
Summary
The epithelial tissue is one of the main types of tissue in the human body. It lines the external body and organ surfaces, providing a permeability barrier that protects against the external environment. The internal cavities of organ systems are lined and compartmentalized into functionally distinct partitions through the selective regulation of ionic and molecular exchange between luminal and interstitial compartments, creating separated tissue microenvironments. Central to this permeability barrier function is the organization of individual epithelial cells into an epithelial sheet (the epithelium) by cell-cell junctions that regulate paracellular movement and the coordinated apico-basal polarization of this sheet into functionally discrete subcellular regions, which facilitate vectorial transcellular transport. A hallmark of epithelial cell-cell junctions is the tight junction (TJ) This structure forms a network of anastomosing intramembranous strands encircling the apico-lateral domain of the epithelial cell, eliminating the paracellular space between adjacent cells. This tight lateral seal is responsible for the epithelial paracellular permeability function [1]
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