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ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

Phua DC, Xu J, Ali SM, Boey A, Gounko NV, Hunziker W - PLoS ONE (2014)

Bottom Line: Through the generation of individual or combined ZO-1 and ZO-2 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.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science Technology and Research (A*STAR), Singapore, Singapore.

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 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 embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

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ExEn organization and permeability is abnormal in EBs lacking ZO-1 and ZO-2.(A) Scanning electron micrographs. ExEn apical surface morphology of WT control and ZO gene-deleted EBs was analyzed by SEM. The apical surface of compacted ExEn cells can be seen covered with dense hair-like microvilli in WT (panel a) and ZO-2-/- (panel c) EBs. ZO-1-/- (panel b) and ZO-1-/- ZO-2-/- (panel d) EB ExEn cells are more loosely packed and bear sparse microvilli formation. Magnification of image in insets. (B) ExEn permeability assay illustration. Intact ExEn of live EBs pre-incubated with anti-BM IgG will not allow penetration of the antibodies to the underlying BM. In a permeability-compromised ExEn, the anti-BM IgG can move across the ExEn paracellularly and bind to the underlying BM. (C) Permeability assay using anti-Lam1+2 pre-incubate. Live WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs at Day-7 (panels a-d) and Day-10 (panels e–h) of culture were pre-incubated for 2 hrs in medium spiked with IgG recognizing the BM component Laminin1+2. The EBs were subsequently washed and fixed, and the embedded sections were then permeabilized and treated with fluorophore-tagged secondary antibodies to visualize the anti-Lam1+2 IgG (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
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pone-0099532-g003: ExEn organization and permeability is abnormal in EBs lacking ZO-1 and ZO-2.(A) Scanning electron micrographs. ExEn apical surface morphology of WT control and ZO gene-deleted EBs was analyzed by SEM. The apical surface of compacted ExEn cells can be seen covered with dense hair-like microvilli in WT (panel a) and ZO-2-/- (panel c) EBs. ZO-1-/- (panel b) and ZO-1-/- ZO-2-/- (panel d) EB ExEn cells are more loosely packed and bear sparse microvilli formation. Magnification of image in insets. (B) ExEn permeability assay illustration. Intact ExEn of live EBs pre-incubated with anti-BM IgG will not allow penetration of the antibodies to the underlying BM. In a permeability-compromised ExEn, the anti-BM IgG can move across the ExEn paracellularly and bind to the underlying BM. (C) Permeability assay using anti-Lam1+2 pre-incubate. Live WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs at Day-7 (panels a-d) and Day-10 (panels e–h) of culture were pre-incubated for 2 hrs in medium spiked with IgG recognizing the BM component Laminin1+2. The EBs were subsequently washed and fixed, and the embedded sections were then permeabilized and treated with fluorophore-tagged secondary antibodies to visualize the anti-Lam1+2 IgG (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.

Mentions: Through the observations of TEM and IF vertical EB sections, we noticed an irregular morphology of the ExEn layer in ZO-1-/- ZO-2-/- EB sections compared to WT control. To explore in detail if the ZO gene-deletions have an effect on ExEn morphology, we visualized the ExEn apical surface using scanning electron microscopy (SEM) at different time points of culture (Fig. 3A). At 5 days of EB culture, WT and ZO-2-/- ExEn cells were arranged in a compact epithelial cobblestone organization and carpeted densely with apical surface microvilli protrusions (Fig. 3A, panels a and c). In contrast, the entire surface of the ExEn layer of ZO-1-/- ZO-2-/- EBs was disorganized and discontiguous, with cell-cell arrangement lacking compactness and boundaries showing obvious gaps. Furthermore, the apical cell surface looked distended and microvilli were sparsely present on this surface (Fig. 3A, panel d). This phenotype was also observed in ZO-1-/- EBs, albeit to a lesser extent, with patches of disorganized cells amidst regularly ordered ones (Fig. 3A, panel b). Interestingly, the irregular surface of ZO-1-/- EB recovered progressively to normalcy by Day-10 of culture, displaying the usual contiguous cobblestone cell arrangement. However, ZO-1-/- ZO-2-/- EBs still exhibited extensive ExEn disorganization throughout the culture period (Fig. S1A and B).


ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

Phua DC, Xu J, Ali SM, Boey A, Gounko NV, Hunziker W - PLoS ONE (2014)

ExEn organization and permeability is abnormal in EBs lacking ZO-1 and ZO-2.(A) Scanning electron micrographs. ExEn apical surface morphology of WT control and ZO gene-deleted EBs was analyzed by SEM. The apical surface of compacted ExEn cells can be seen covered with dense hair-like microvilli in WT (panel a) and ZO-2-/- (panel c) EBs. ZO-1-/- (panel b) and ZO-1-/- ZO-2-/- (panel d) EB ExEn cells are more loosely packed and bear sparse microvilli formation. Magnification of image in insets. (B) ExEn permeability assay illustration. Intact ExEn of live EBs pre-incubated with anti-BM IgG will not allow penetration of the antibodies to the underlying BM. In a permeability-compromised ExEn, the anti-BM IgG can move across the ExEn paracellularly and bind to the underlying BM. (C) Permeability assay using anti-Lam1+2 pre-incubate. Live WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs at Day-7 (panels a-d) and Day-10 (panels e–h) of culture were pre-incubated for 2 hrs in medium spiked with IgG recognizing the BM component Laminin1+2. The EBs were subsequently washed and fixed, and the embedded sections were then permeabilized and treated with fluorophore-tagged secondary antibodies to visualize the anti-Lam1+2 IgG (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048262&req=5

pone-0099532-g003: ExEn organization and permeability is abnormal in EBs lacking ZO-1 and ZO-2.(A) Scanning electron micrographs. ExEn apical surface morphology of WT control and ZO gene-deleted EBs was analyzed by SEM. The apical surface of compacted ExEn cells can be seen covered with dense hair-like microvilli in WT (panel a) and ZO-2-/- (panel c) EBs. ZO-1-/- (panel b) and ZO-1-/- ZO-2-/- (panel d) EB ExEn cells are more loosely packed and bear sparse microvilli formation. Magnification of image in insets. (B) ExEn permeability assay illustration. Intact ExEn of live EBs pre-incubated with anti-BM IgG will not allow penetration of the antibodies to the underlying BM. In a permeability-compromised ExEn, the anti-BM IgG can move across the ExEn paracellularly and bind to the underlying BM. (C) Permeability assay using anti-Lam1+2 pre-incubate. Live WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs at Day-7 (panels a-d) and Day-10 (panels e–h) of culture were pre-incubated for 2 hrs in medium spiked with IgG recognizing the BM component Laminin1+2. The EBs were subsequently washed and fixed, and the embedded sections were then permeabilized and treated with fluorophore-tagged secondary antibodies to visualize the anti-Lam1+2 IgG (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
Mentions: Through the observations of TEM and IF vertical EB sections, we noticed an irregular morphology of the ExEn layer in ZO-1-/- ZO-2-/- EB sections compared to WT control. To explore in detail if the ZO gene-deletions have an effect on ExEn morphology, we visualized the ExEn apical surface using scanning electron microscopy (SEM) at different time points of culture (Fig. 3A). At 5 days of EB culture, WT and ZO-2-/- ExEn cells were arranged in a compact epithelial cobblestone organization and carpeted densely with apical surface microvilli protrusions (Fig. 3A, panels a and c). In contrast, the entire surface of the ExEn layer of ZO-1-/- ZO-2-/- EBs was disorganized and discontiguous, with cell-cell arrangement lacking compactness and boundaries showing obvious gaps. Furthermore, the apical cell surface looked distended and microvilli were sparsely present on this surface (Fig. 3A, panel d). This phenotype was also observed in ZO-1-/- EBs, albeit to a lesser extent, with patches of disorganized cells amidst regularly ordered ones (Fig. 3A, panel b). Interestingly, the irregular surface of ZO-1-/- EB recovered progressively to normalcy by Day-10 of culture, displaying the usual contiguous cobblestone cell arrangement. However, ZO-1-/- ZO-2-/- EBs still exhibited extensive ExEn disorganization throughout the culture period (Fig. S1A and B).

Bottom Line: Through the generation of individual or combined ZO-1 and ZO-2 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.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Cell Biology Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science Technology and Research (A*STAR), Singapore, Singapore.

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 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 embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.

Show MeSH
Related in: MedlinePlus