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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

PODXL expression and Ezrin localization at the ExEn is aberrant when ZO-1 and ZO-2 is deleted.(A) Immunofluorescence staining of PODXL. EB cryosections from Day-5 (panels a–h) and -9 (panels i–p) cultures were immunostained for PODXL (green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) PODXL expression. Protein expression of PODXL at Day-4 and -6 was determined by immunoblot. 100 (immature glycosylated), 140 (mature glycosylated) and 250 (dimer) kD bands represent the various post-translationally modified forms of PODXL. GAPDH was used as a lysate loading control (panel a). PODXL transcript level was analyzed by semi-quantitative RT-PCR. Reverse transcribed cDNA was amplified with specific primer sets at optimized cycle numbers (indicated on right side of panels). Ezrin and Pals1 were selected as epithelia polarized controls. GAPDH amplification served as a control for equal RNA input (panel b). Quantitative real-time PCR was also employed to validate PODXL transcript expression. Expression levels are presented as average fold-change of three separate experiments normalized to GAPDH and relative to WT control (panel c). (C) Immunofluorescence staining of Ezrin. Cryosections of EBs harvested at Day-4 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l) of culture were immunostained with antibodies against Ezrin (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
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pone-0099532-g005: PODXL expression and Ezrin localization at the ExEn is aberrant when ZO-1 and ZO-2 is deleted.(A) Immunofluorescence staining of PODXL. EB cryosections from Day-5 (panels a–h) and -9 (panels i–p) cultures were immunostained for PODXL (green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) PODXL expression. Protein expression of PODXL at Day-4 and -6 was determined by immunoblot. 100 (immature glycosylated), 140 (mature glycosylated) and 250 (dimer) kD bands represent the various post-translationally modified forms of PODXL. GAPDH was used as a lysate loading control (panel a). PODXL transcript level was analyzed by semi-quantitative RT-PCR. Reverse transcribed cDNA was amplified with specific primer sets at optimized cycle numbers (indicated on right side of panels). Ezrin and Pals1 were selected as epithelia polarized controls. GAPDH amplification served as a control for equal RNA input (panel b). Quantitative real-time PCR was also employed to validate PODXL transcript expression. Expression levels are presented as average fold-change of three separate experiments normalized to GAPDH and relative to WT control (panel c). (C) Immunofluorescence staining of Ezrin. Cryosections of EBs harvested at Day-4 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l) of culture were immunostained with antibodies against Ezrin (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.

Mentions: Podocalyxin (PODXL) is a single-pass transmembrane sialoglycoprotein that was first discovered in specialized epithelial cells of the renal glomerulus (podocytes) [32]. It is localized at the apical membrane of polarized epithelial cells and has a heavily sialylated ectodomain and a cytosolic tail that associates indirectly with actin filaments [33]. Since PODXL positively regulates the formation of microvilli [34] and the apical domain [35] in epithelial cells, we investigated if ZO deficiency had an effect on this protein. EBs were monitored for PODXL expression and localization continually over a 9 day period of culture by immunofluorescence microscopy. At Day-5, WT and ZO-2-/- EBs (Fig. 5A, panels a and e; panels c and g, respectively) exhibited an accumulation of PODXL at the apical membrane of the entire ExEn layer. This staining could also be seen on the apical membrane of the primitive ectoderm (PrEc) surrounding nascent cavities at the EB interior. However, in ZO-1-/- EBs (Fig. 5A, panels b and f), apical membrane PODXL staining was not continuous along the entire ExEn layer, with some areas of the ExEn lacking PODXL. This anomaly was even more pronounced with ZO-1-/- ZO-2-/- EBs (Fig. 5A, panels d and h), where apical membrane PODXL concentration was fragmented and faint throughout the ExEn layer. As with the restoration to normalcy of the ExEn cell organization and permeability function reported earlier, the ZO-1-/- ExEn layer displayed a continuous apical membrane staining at later time points of culture, as depicted at Day-9 (Fig. 5A, panels j and h). This staining was indistinguishable from the normal PODXL apical membrane enrichment in WT and ZO-2-/- ExEn at Day-9 (Fig. 5A, panels i and m; panels k and o, respectively). As expected, apical membrane PODXL staining of ZO-1-/- ZO-2-/- ExEn remained faint and discontinuous even at Day-9 (Fig. 5A, panels l and p), indicating an inability for recovery to normalcy.


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)

PODXL expression and Ezrin localization at the ExEn is aberrant when ZO-1 and ZO-2 is deleted.(A) Immunofluorescence staining of PODXL. EB cryosections from Day-5 (panels a–h) and -9 (panels i–p) cultures were immunostained for PODXL (green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) PODXL expression. Protein expression of PODXL at Day-4 and -6 was determined by immunoblot. 100 (immature glycosylated), 140 (mature glycosylated) and 250 (dimer) kD bands represent the various post-translationally modified forms of PODXL. GAPDH was used as a lysate loading control (panel a). PODXL transcript level was analyzed by semi-quantitative RT-PCR. Reverse transcribed cDNA was amplified with specific primer sets at optimized cycle numbers (indicated on right side of panels). Ezrin and Pals1 were selected as epithelia polarized controls. GAPDH amplification served as a control for equal RNA input (panel b). Quantitative real-time PCR was also employed to validate PODXL transcript expression. Expression levels are presented as average fold-change of three separate experiments normalized to GAPDH and relative to WT control (panel c). (C) Immunofluorescence staining of Ezrin. Cryosections of EBs harvested at Day-4 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l) of culture were immunostained with antibodies against Ezrin (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
© Copyright Policy
Related In: Results  -  Collection

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pone-0099532-g005: PODXL expression and Ezrin localization at the ExEn is aberrant when ZO-1 and ZO-2 is deleted.(A) Immunofluorescence staining of PODXL. EB cryosections from Day-5 (panels a–h) and -9 (panels i–p) cultures were immunostained for PODXL (green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) PODXL expression. Protein expression of PODXL at Day-4 and -6 was determined by immunoblot. 100 (immature glycosylated), 140 (mature glycosylated) and 250 (dimer) kD bands represent the various post-translationally modified forms of PODXL. GAPDH was used as a lysate loading control (panel a). PODXL transcript level was analyzed by semi-quantitative RT-PCR. Reverse transcribed cDNA was amplified with specific primer sets at optimized cycle numbers (indicated on right side of panels). Ezrin and Pals1 were selected as epithelia polarized controls. GAPDH amplification served as a control for equal RNA input (panel b). Quantitative real-time PCR was also employed to validate PODXL transcript expression. Expression levels are presented as average fold-change of three separate experiments normalized to GAPDH and relative to WT control (panel c). (C) Immunofluorescence staining of Ezrin. Cryosections of EBs harvested at Day-4 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l) of culture were immunostained with antibodies against Ezrin (red color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets.
Mentions: Podocalyxin (PODXL) is a single-pass transmembrane sialoglycoprotein that was first discovered in specialized epithelial cells of the renal glomerulus (podocytes) [32]. It is localized at the apical membrane of polarized epithelial cells and has a heavily sialylated ectodomain and a cytosolic tail that associates indirectly with actin filaments [33]. Since PODXL positively regulates the formation of microvilli [34] and the apical domain [35] in epithelial cells, we investigated if ZO deficiency had an effect on this protein. EBs were monitored for PODXL expression and localization continually over a 9 day period of culture by immunofluorescence microscopy. At Day-5, WT and ZO-2-/- EBs (Fig. 5A, panels a and e; panels c and g, respectively) exhibited an accumulation of PODXL at the apical membrane of the entire ExEn layer. This staining could also be seen on the apical membrane of the primitive ectoderm (PrEc) surrounding nascent cavities at the EB interior. However, in ZO-1-/- EBs (Fig. 5A, panels b and f), apical membrane PODXL staining was not continuous along the entire ExEn layer, with some areas of the ExEn lacking PODXL. This anomaly was even more pronounced with ZO-1-/- ZO-2-/- EBs (Fig. 5A, panels d and h), where apical membrane PODXL concentration was fragmented and faint throughout the ExEn layer. As with the restoration to normalcy of the ExEn cell organization and permeability function reported earlier, the ZO-1-/- ExEn layer displayed a continuous apical membrane staining at later time points of culture, as depicted at Day-9 (Fig. 5A, panels j and h). This staining was indistinguishable from the normal PODXL apical membrane enrichment in WT and ZO-2-/- ExEn at Day-9 (Fig. 5A, panels i and m; panels k and o, respectively). As expected, apical membrane PODXL staining of ZO-1-/- ZO-2-/- ExEn remained faint and discontinuous even at Day-9 (Fig. 5A, panels l and p), indicating an inability for recovery to normalcy.

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