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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

EB development and TJ formation.(A) Schematic diagram of EB development. The development of EBs starts with the aggregation of ESCs into suspended spheroids of inner cell mass (ICM) and parallels the development of the mouse blastocyst ICM. By day 3 of culture, the outer cells differentiate into a circumferential epithelium known as the primitive endoderm (PrEn). The apical and basal domains of the PrEn face the exterior environment and interior ICM core of the EB respectively. The EBs at this point are called simple EBs. Progressively from day 4 onwards, the PrEn basally secretes key ECM components Laminins and Collagen IV to form a basement membrane (BM). The PrEn also further differentiates in the visceral endoderm (VEn). In conjunction with this, the interior ICM differentiates into the epiblast. By day 5 or 6, the epiblast in contact with the BM in turn differentiates into the primitive ectoderm (PrEc) or epiblast epithelium. The rest of the epiblast not in contact with the BM will undergo apoptosis. The apoptotic bodies are removed by autophagy-initiated phagocytosis, leaving a progressively enlarging lumen or Proamniotic-like cavity (PAC) surrounded by the apical domain of the PrEc and the basal BM. EBs at this stage are called cystic EBs and equivalent to the egg cylinder stage of the mouse peri-implantation embryo just before gastrulation. (B) Immunofluorescence staining of ZO-1 and ZO-2 in Day-5 or -6 EB cryosections. WT (panels a and e), ZO-1-/- (panels b and f), ZO-2-/- (panels c and g) and ZO-1-/- ZO-2-/- (panels d and h) EBs were stained with antibodies to ZO-1 (panels a-d, green color) or ZO-2 (panels e-h, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’. (C) Transmission electron micrographs. TJ complex at the apico-lateral membrane of Day-9 EB ExEn was visualized by TEM as electron-dense material (indicated by arrows in magnification of inset) in WT (panels a and e), ZO-1-/- (panels b and f) and ZO-2-/- (panels c and g) EBs. This was absent in ZO-1-/- ZO-2-/- EBs (panels d and h, arrowheads in magnification of inset). (D) Immunofluorescence staining of Cldn-6 in Day-9 EB cryosections. WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs were stained with antibodies to Cldn-6 (panels a–d, green 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-g002: EB development and TJ formation.(A) Schematic diagram of EB development. The development of EBs starts with the aggregation of ESCs into suspended spheroids of inner cell mass (ICM) and parallels the development of the mouse blastocyst ICM. By day 3 of culture, the outer cells differentiate into a circumferential epithelium known as the primitive endoderm (PrEn). The apical and basal domains of the PrEn face the exterior environment and interior ICM core of the EB respectively. The EBs at this point are called simple EBs. Progressively from day 4 onwards, the PrEn basally secretes key ECM components Laminins and Collagen IV to form a basement membrane (BM). The PrEn also further differentiates in the visceral endoderm (VEn). In conjunction with this, the interior ICM differentiates into the epiblast. By day 5 or 6, the epiblast in contact with the BM in turn differentiates into the primitive ectoderm (PrEc) or epiblast epithelium. The rest of the epiblast not in contact with the BM will undergo apoptosis. The apoptotic bodies are removed by autophagy-initiated phagocytosis, leaving a progressively enlarging lumen or Proamniotic-like cavity (PAC) surrounded by the apical domain of the PrEc and the basal BM. EBs at this stage are called cystic EBs and equivalent to the egg cylinder stage of the mouse peri-implantation embryo just before gastrulation. (B) Immunofluorescence staining of ZO-1 and ZO-2 in Day-5 or -6 EB cryosections. WT (panels a and e), ZO-1-/- (panels b and f), ZO-2-/- (panels c and g) and ZO-1-/- ZO-2-/- (panels d and h) EBs were stained with antibodies to ZO-1 (panels a-d, green color) or ZO-2 (panels e-h, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’. (C) Transmission electron micrographs. TJ complex at the apico-lateral membrane of Day-9 EB ExEn was visualized by TEM as electron-dense material (indicated by arrows in magnification of inset) in WT (panels a and e), ZO-1-/- (panels b and f) and ZO-2-/- (panels c and g) EBs. This was absent in ZO-1-/- ZO-2-/- EBs (panels d and h, arrowheads in magnification of inset). (D) Immunofluorescence staining of Cldn-6 in Day-9 EB cryosections. WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs were stained with antibodies to Cldn-6 (panels a–d, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.

Mentions: To investigate the functional roles of ZO-1 and ZO-2 in epithelial morphogenesis and de novo TJ biogenesis, mESCs were cultured into EBs. The development of EBs is illustrated and described in Fig. 2A.


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 development and TJ formation.(A) Schematic diagram of EB development. The development of EBs starts with the aggregation of ESCs into suspended spheroids of inner cell mass (ICM) and parallels the development of the mouse blastocyst ICM. By day 3 of culture, the outer cells differentiate into a circumferential epithelium known as the primitive endoderm (PrEn). The apical and basal domains of the PrEn face the exterior environment and interior ICM core of the EB respectively. The EBs at this point are called simple EBs. Progressively from day 4 onwards, the PrEn basally secretes key ECM components Laminins and Collagen IV to form a basement membrane (BM). The PrEn also further differentiates in the visceral endoderm (VEn). In conjunction with this, the interior ICM differentiates into the epiblast. By day 5 or 6, the epiblast in contact with the BM in turn differentiates into the primitive ectoderm (PrEc) or epiblast epithelium. The rest of the epiblast not in contact with the BM will undergo apoptosis. The apoptotic bodies are removed by autophagy-initiated phagocytosis, leaving a progressively enlarging lumen or Proamniotic-like cavity (PAC) surrounded by the apical domain of the PrEc and the basal BM. EBs at this stage are called cystic EBs and equivalent to the egg cylinder stage of the mouse peri-implantation embryo just before gastrulation. (B) Immunofluorescence staining of ZO-1 and ZO-2 in Day-5 or -6 EB cryosections. WT (panels a and e), ZO-1-/- (panels b and f), ZO-2-/- (panels c and g) and ZO-1-/- ZO-2-/- (panels d and h) EBs were stained with antibodies to ZO-1 (panels a-d, green color) or ZO-2 (panels e-h, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’. (C) Transmission electron micrographs. TJ complex at the apico-lateral membrane of Day-9 EB ExEn was visualized by TEM as electron-dense material (indicated by arrows in magnification of inset) in WT (panels a and e), ZO-1-/- (panels b and f) and ZO-2-/- (panels c and g) EBs. This was absent in ZO-1-/- ZO-2-/- EBs (panels d and h, arrowheads in magnification of inset). (D) Immunofluorescence staining of Cldn-6 in Day-9 EB cryosections. WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs were stained with antibodies to Cldn-6 (panels a–d, green 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

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

pone-0099532-g002: EB development and TJ formation.(A) Schematic diagram of EB development. The development of EBs starts with the aggregation of ESCs into suspended spheroids of inner cell mass (ICM) and parallels the development of the mouse blastocyst ICM. By day 3 of culture, the outer cells differentiate into a circumferential epithelium known as the primitive endoderm (PrEn). The apical and basal domains of the PrEn face the exterior environment and interior ICM core of the EB respectively. The EBs at this point are called simple EBs. Progressively from day 4 onwards, the PrEn basally secretes key ECM components Laminins and Collagen IV to form a basement membrane (BM). The PrEn also further differentiates in the visceral endoderm (VEn). In conjunction with this, the interior ICM differentiates into the epiblast. By day 5 or 6, the epiblast in contact with the BM in turn differentiates into the primitive ectoderm (PrEc) or epiblast epithelium. The rest of the epiblast not in contact with the BM will undergo apoptosis. The apoptotic bodies are removed by autophagy-initiated phagocytosis, leaving a progressively enlarging lumen or Proamniotic-like cavity (PAC) surrounded by the apical domain of the PrEc and the basal BM. EBs at this stage are called cystic EBs and equivalent to the egg cylinder stage of the mouse peri-implantation embryo just before gastrulation. (B) Immunofluorescence staining of ZO-1 and ZO-2 in Day-5 or -6 EB cryosections. WT (panels a and e), ZO-1-/- (panels b and f), ZO-2-/- (panels c and g) and ZO-1-/- ZO-2-/- (panels d and h) EBs were stained with antibodies to ZO-1 (panels a-d, green color) or ZO-2 (panels e-h, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’. (C) Transmission electron micrographs. TJ complex at the apico-lateral membrane of Day-9 EB ExEn was visualized by TEM as electron-dense material (indicated by arrows in magnification of inset) in WT (panels a and e), ZO-1-/- (panels b and f) and ZO-2-/- (panels c and g) EBs. This was absent in ZO-1-/- ZO-2-/- EBs (panels d and h, arrowheads in magnification of inset). (D) Immunofluorescence staining of Cldn-6 in Day-9 EB cryosections. WT, ZO-1-/-, ZO-2-/- and ZO-1-/- ZO-2-/- EBs were stained with antibodies to Cldn-6 (panels a–d, green color). Nuclei are labeled with DAPI (blue color). Magnification of image in insets. ExEn is indicated here as ‘en’.
Mentions: To investigate the functional roles of ZO-1 and ZO-2 in epithelial morphogenesis and de novo TJ biogenesis, mESCs were cultured into EBs. The development of EBs is illustrated and described in Fig. 2A.

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