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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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Depletion of ZO-1 and ZO-2 is associated with aberrant basement membrane deposition and irregular cavitation.(A) Immunofluorescence staining of Nidogen. EB cryosections from Day-9 cultures (panels a–d) were immunostained for the BM component Nidogen (red color, arrow). ExEn and BM are indicated by arrowhead and arrow respectively. Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) Immunofluorescence staining of PrEc. The immunodetection of Integrin α6 was used as a specific marker for PrEc visualization in Day-10 EB cultures (panels a–d, red color). Nuclei are labeled with DAPI (blue color). PrEc is indicated here as ‘ec’. (C) Phase-contrast microscopy. Cavity development in live EBs was tracked by phase-contrast imaging on Day-3 (panels a–d), Day-5 (panels e–h), Day-7 (panels i–l) and Day-9 (panels m–p) of culture. (D) H&E histological staining. PFA-fixed and paraffin-embedded EB sections were treated with H&E stains to visualize the gross structure of the EBs. EB histology was analyzed at Day-5 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l). (E) TUNEL staining of apoptotic cells. Cryosections of EBs at Day-9 culture were immunostained with Nidogen (Nid) to demarcate the BM boundary of cavities (panels a,d,g,j, green color). TUNEL-positive apoptotic cells were visualized in red color (panels b,e,h,k). Merged images (panels c,f,i,l). Nuclei are labeled with DAPI (blue color).
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pone-0099532-g006: Depletion of ZO-1 and ZO-2 is associated with aberrant basement membrane deposition and irregular cavitation.(A) Immunofluorescence staining of Nidogen. EB cryosections from Day-9 cultures (panels a–d) were immunostained for the BM component Nidogen (red color, arrow). ExEn and BM are indicated by arrowhead and arrow respectively. Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) Immunofluorescence staining of PrEc. The immunodetection of Integrin α6 was used as a specific marker for PrEc visualization in Day-10 EB cultures (panels a–d, red color). Nuclei are labeled with DAPI (blue color). PrEc is indicated here as ‘ec’. (C) Phase-contrast microscopy. Cavity development in live EBs was tracked by phase-contrast imaging on Day-3 (panels a–d), Day-5 (panels e–h), Day-7 (panels i–l) and Day-9 (panels m–p) of culture. (D) H&E histological staining. PFA-fixed and paraffin-embedded EB sections were treated with H&E stains to visualize the gross structure of the EBs. EB histology was analyzed at Day-5 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l). (E) TUNEL staining of apoptotic cells. Cryosections of EBs at Day-9 culture were immunostained with Nidogen (Nid) to demarcate the BM boundary of cavities (panels a,d,g,j, green color). TUNEL-positive apoptotic cells were visualized in red color (panels b,e,h,k). Merged images (panels c,f,i,l). Nuclei are labeled with DAPI (blue color).

Mentions: A hallmark of epithelial morphogenesis is the deposition of a basal basement membrane (BM). In EBs, key BM components like Laminins and Collagen IV are secreted by the ExEn and deposited as a basal underlying band [43]. As the ExEn layer is disorganized in the ZO-1-/- ZO-2-/- EBs, we investigated if this had an effect on the deposition of the BM. The BM consists of various components, including Laminins, Collagen, Perlecan and Nidogen [44], with Laminins playing a key role in assembly of the BM [45]. Immunofluorescence staining of Nidogen (Nid) (Fig. 6A), and Perlecan, Collagen IV and Laminin 1+2 (Fig. S4), was done on Day-9 and Day-12 cultures, respectively, when BM deposition in WT control EBs was complete. At Day-9, Nidogen (denoted as ‘bm’ in image) is visible as a continuous band underlying the ExEn in WT, ZO-1-/- and ZO-2-/- EBs (Fig. 6A, panels a–c). In contrast, in ZO-1-/- ZO-2-/- EBs, the BM was instead irregularly deposited and looked discontinuous and fragmented (Fig. 6A, panel d), probably due to the disorganization of the ExEn. This same pattern of BM deposition was also observed in Day-12 EB cultures stained for the other BM components (Fig. S4).


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)

Depletion of ZO-1 and ZO-2 is associated with aberrant basement membrane deposition and irregular cavitation.(A) Immunofluorescence staining of Nidogen. EB cryosections from Day-9 cultures (panels a–d) were immunostained for the BM component Nidogen (red color, arrow). ExEn and BM are indicated by arrowhead and arrow respectively. Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) Immunofluorescence staining of PrEc. The immunodetection of Integrin α6 was used as a specific marker for PrEc visualization in Day-10 EB cultures (panels a–d, red color). Nuclei are labeled with DAPI (blue color). PrEc is indicated here as ‘ec’. (C) Phase-contrast microscopy. Cavity development in live EBs was tracked by phase-contrast imaging on Day-3 (panels a–d), Day-5 (panels e–h), Day-7 (panels i–l) and Day-9 (panels m–p) of culture. (D) H&E histological staining. PFA-fixed and paraffin-embedded EB sections were treated with H&E stains to visualize the gross structure of the EBs. EB histology was analyzed at Day-5 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l). (E) TUNEL staining of apoptotic cells. Cryosections of EBs at Day-9 culture were immunostained with Nidogen (Nid) to demarcate the BM boundary of cavities (panels a,d,g,j, green color). TUNEL-positive apoptotic cells were visualized in red color (panels b,e,h,k). Merged images (panels c,f,i,l). Nuclei are labeled with DAPI (blue color).
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pone-0099532-g006: Depletion of ZO-1 and ZO-2 is associated with aberrant basement membrane deposition and irregular cavitation.(A) Immunofluorescence staining of Nidogen. EB cryosections from Day-9 cultures (panels a–d) were immunostained for the BM component Nidogen (red color, arrow). ExEn and BM are indicated by arrowhead and arrow respectively. Nuclei are labeled with DAPI (blue color). Magnification of image in insets. (B) Immunofluorescence staining of PrEc. The immunodetection of Integrin α6 was used as a specific marker for PrEc visualization in Day-10 EB cultures (panels a–d, red color). Nuclei are labeled with DAPI (blue color). PrEc is indicated here as ‘ec’. (C) Phase-contrast microscopy. Cavity development in live EBs was tracked by phase-contrast imaging on Day-3 (panels a–d), Day-5 (panels e–h), Day-7 (panels i–l) and Day-9 (panels m–p) of culture. (D) H&E histological staining. PFA-fixed and paraffin-embedded EB sections were treated with H&E stains to visualize the gross structure of the EBs. EB histology was analyzed at Day-5 (panels a–d), Day-7 (panels e–h) and Day-9 (panels i–l). (E) TUNEL staining of apoptotic cells. Cryosections of EBs at Day-9 culture were immunostained with Nidogen (Nid) to demarcate the BM boundary of cavities (panels a,d,g,j, green color). TUNEL-positive apoptotic cells were visualized in red color (panels b,e,h,k). Merged images (panels c,f,i,l). Nuclei are labeled with DAPI (blue color).
Mentions: A hallmark of epithelial morphogenesis is the deposition of a basal basement membrane (BM). In EBs, key BM components like Laminins and Collagen IV are secreted by the ExEn and deposited as a basal underlying band [43]. As the ExEn layer is disorganized in the ZO-1-/- ZO-2-/- EBs, we investigated if this had an effect on the deposition of the BM. The BM consists of various components, including Laminins, Collagen, Perlecan and Nidogen [44], with Laminins playing a key role in assembly of the BM [45]. Immunofluorescence staining of Nidogen (Nid) (Fig. 6A), and Perlecan, Collagen IV and Laminin 1+2 (Fig. S4), was done on Day-9 and Day-12 cultures, respectively, when BM deposition in WT control EBs was complete. At Day-9, Nidogen (denoted as ‘bm’ in image) is visible as a continuous band underlying the ExEn in WT, ZO-1-/- and ZO-2-/- EBs (Fig. 6A, panels a–c). In contrast, in ZO-1-/- ZO-2-/- EBs, the BM was instead irregularly deposited and looked discontinuous and fragmented (Fig. 6A, panel d), probably due to the disorganization of the ExEn. This same pattern of BM deposition was also observed in Day-12 EB cultures stained for the other BM components (Fig. S4).

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