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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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EB ExEn differentiation is not affected by ZO-1 and/or ZO-2 deletion.(A) Semi-quantitative RT-PCR of Day-4 EB. Reverse transcribed cDNA was amplified with specific GATA-4 and -6 primer sets at optimized cycle numbers (indicated on right side of panels). GAPDH amplification served as a control for equal RNA input. (B) Immunofluorescence staining of EB ExEn. Fixed and permeabilized EB cryosections were treated with antibodies immunoreactive to Dab2 (panels a-d) and Keratin-8 (Troma-1) (panels e-h) and visualized (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.
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pone-0099532-g004: EB ExEn differentiation is not affected by ZO-1 and/or ZO-2 deletion.(A) Semi-quantitative RT-PCR of Day-4 EB. Reverse transcribed cDNA was amplified with specific GATA-4 and -6 primer sets at optimized cycle numbers (indicated on right side of panels). GAPDH amplification served as a control for equal RNA input. (B) Immunofluorescence staining of EB ExEn. Fixed and permeabilized EB cryosections were treated with antibodies immunoreactive to Dab2 (panels a-d) and Keratin-8 (Troma-1) (panels e-h) and visualized (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.

Mentions: The presence of GATA-4 and -6 mRNA transcripts in EBs of Day-4 culture were investigated by RT-PCR (Fig. 4A). Day 4 EBs were chosen for this semi-quantitative study as the PrEn would have differentiated by this time. After optimizing the PCR cycle number to coincide with the exponential phase of amplification, the amplicon intensity between WT and the three mutants for both GATA-4 and -6 amplifications were indistinguishable. The GATA-6 downstream target gene, Dab2 was next tested for expression at the protein level in the ExEn by immunostaining (Fig. 4B, panels a–d). Dab2 was detected in the cytoplasm of the ExEn of all three ZO mutants and the WT control EBs. Similarly, Keratin-8 (Troma-1) was present in all EB types (Fig. 4B panels e–h), thus indicating that the ExEn layer is indeed differentiated and is of epithelial cell-type in the EBs lacking ZO proteins.


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)

EB ExEn differentiation is not affected by ZO-1 and/or ZO-2 deletion.(A) Semi-quantitative RT-PCR of Day-4 EB. Reverse transcribed cDNA was amplified with specific GATA-4 and -6 primer sets at optimized cycle numbers (indicated on right side of panels). GAPDH amplification served as a control for equal RNA input. (B) Immunofluorescence staining of EB ExEn. Fixed and permeabilized EB cryosections were treated with antibodies immunoreactive to Dab2 (panels a-d) and Keratin-8 (Troma-1) (panels e-h) and visualized (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099532-g004: EB ExEn differentiation is not affected by ZO-1 and/or ZO-2 deletion.(A) Semi-quantitative RT-PCR of Day-4 EB. Reverse transcribed cDNA was amplified with specific GATA-4 and -6 primer sets at optimized cycle numbers (indicated on right side of panels). GAPDH amplification served as a control for equal RNA input. (B) Immunofluorescence staining of EB ExEn. Fixed and permeabilized EB cryosections were treated with antibodies immunoreactive to Dab2 (panels a-d) and Keratin-8 (Troma-1) (panels e-h) and visualized (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.
Mentions: The presence of GATA-4 and -6 mRNA transcripts in EBs of Day-4 culture were investigated by RT-PCR (Fig. 4A). Day 4 EBs were chosen for this semi-quantitative study as the PrEn would have differentiated by this time. After optimizing the PCR cycle number to coincide with the exponential phase of amplification, the amplicon intensity between WT and the three mutants for both GATA-4 and -6 amplifications were indistinguishable. The GATA-6 downstream target gene, Dab2 was next tested for expression at the protein level in the ExEn by immunostaining (Fig. 4B, panels a–d). Dab2 was detected in the cytoplasm of the ExEn of all three ZO mutants and the WT control EBs. Similarly, Keratin-8 (Troma-1) was present in all EB types (Fig. 4B panels e–h), thus indicating that the ExEn layer is indeed differentiated and is of epithelial cell-type in the EBs lacking ZO proteins.

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