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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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Related in: MedlinePlus

Targeting of the ZO-1 and ZO-2 locus and genotyping.(A) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. (B) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). (C) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.
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pone-0099532-g001: Targeting of the ZO-1 and ZO-2 locus and genotyping.(A) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. (B) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). (C) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.

Mentions: The ZO-1 (Fig. 1A, panel a) or ZO-2 (Fig. 1A, panel b) gene locus was targeted in W4 mESCs with a β-galactosidase gene (LacZ) knock-in targeting vector using the strategy as previously described [19][26] and illustrated in Fig. 1A. Homozygous ZO-1 and ZO-2 double gene-deleted mESCs (ZO-1-/- ZO-2-/-) were generated from the targeting of the ZO-2 locus of two independent ZO-1-/- mESC clones. Targeted mESCs were selected in G418 and screened for homologous recombination at the ZO-1 and/or ZO-2 gene locus by Southern blot hybridization of ScaI-digested genomic DNA with gene-specific 5′ Arm probes (Fig. 1B, panels a and b). The 5′-Arm ZO-1 probe detected a 10 kb and 14.8 kb band corresponding to the ZO-1 WT and mutant alleles respectively. Only the ZO-1-/- and ZO-1-/- ZO-2-/- mESC ScaI-digests 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). The ZO-2 WT and mutant alleles were detected as 6.7 kb and 11.5 kb bands by the 5′-Arm ZO-2 probe. ZO-2 mutant but not WT alleles were identified in ZO-2-/- and ZO-1-/-ZO-2-/- mESCs. Conversely, only ZO-2 WT alleles were observed in WT and ZO-1-/- mESCs (Fig. 1B, panel b). Western blot analysis validated the genotype results, showing an absence of both ZO-1 and ZO-2 proteins in ZO-1-/- ZO-2-/- mESCs, but absence of only ZO-1 protein in ZO-1-/- and ZO-2 protein in ZO-2-/- mESCs (Fig. 1B, panel c), thus indicating the successful targeting of the ZO-1 and/or ZO-2 genes.


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)

Targeting of the ZO-1 and ZO-2 locus and genotyping.(A) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. (B) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). (C) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099532-g001: Targeting of the ZO-1 and ZO-2 locus and genotyping.(A) Targeting strategy. Schematic representation of the genomic loci restriction maps of ZO-1 showing exon 1 (panel a) and ZO-2 showing exons 2–3 (panel b) in yellow boxes with the initiation ATG. Through the in-frame insertion of a LacZ gene (green box) and a loxP-flanked (purple circle) Neo cassette (white box) immediately downstream of the ATG codon, the ZO-1 and ZO-2 allele-targeting constructs were designed to delete the entire ZO-1 exon 1 and part of the downstream intron; and part of ZO-2 exon 2 respectively. The red bar indicates the position of probe hybridization for Southern blot analysis. (B) Genotypic analysis by Southern blotting. ScaI-digested genomic DNA of selected mESC clones were hybridized with a DIG-labelled 5′ genomic DNA probe for the identification of homologous recombinants. 10 kb and 14.8 kb probe-hybridized fragments correspond to WT and targeted allele of ZO-1 locus respectively (panel a), whereas WT and targeted allele of ZO-2 locus are respectively represented by 6.7 kb and 11.5 kb fragments (panel b). (C) Protein expression analysis. mESC lysates were subjected to immunoblotting with anti-ZO-1 and anti-ZO-2 antibodies. The presence of ZO-1 and ZO-2 protein is indicated by 220 kD and 160 kD bands respectively. GAPDH served as a control for equal lysate input.
Mentions: The ZO-1 (Fig. 1A, panel a) or ZO-2 (Fig. 1A, panel b) gene locus was targeted in W4 mESCs with a β-galactosidase gene (LacZ) knock-in targeting vector using the strategy as previously described [19][26] and illustrated in Fig. 1A. Homozygous ZO-1 and ZO-2 double gene-deleted mESCs (ZO-1-/- ZO-2-/-) were generated from the targeting of the ZO-2 locus of two independent ZO-1-/- mESC clones. Targeted mESCs were selected in G418 and screened for homologous recombination at the ZO-1 and/or ZO-2 gene locus by Southern blot hybridization of ScaI-digested genomic DNA with gene-specific 5′ Arm probes (Fig. 1B, panels a and b). The 5′-Arm ZO-1 probe detected a 10 kb and 14.8 kb band corresponding to the ZO-1 WT and mutant alleles respectively. Only the ZO-1-/- and ZO-1-/- ZO-2-/- mESC ScaI-digests 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). The ZO-2 WT and mutant alleles were detected as 6.7 kb and 11.5 kb bands by the 5′-Arm ZO-2 probe. ZO-2 mutant but not WT alleles were identified in ZO-2-/- and ZO-1-/-ZO-2-/- mESCs. Conversely, only ZO-2 WT alleles were observed in WT and ZO-1-/- mESCs (Fig. 1B, panel b). Western blot analysis validated the genotype results, showing an absence of both ZO-1 and ZO-2 proteins in ZO-1-/- ZO-2-/- mESCs, but absence of only ZO-1 protein in ZO-1-/- and ZO-2 protein in ZO-2-/- mESCs (Fig. 1B, panel c), thus indicating the successful targeting of the ZO-1 and/or ZO-2 genes.

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