Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development - PubMed (original) (raw)

Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development

Jianliang Xu et al. Mol Cell Biol. 2008 Mar.

Abstract

ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3 knockout mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2(-)(/)(-) embryos show decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5 compared to wild-type embryos. The asymmetric distribution of prominin and E-cadherin to the apical and lateral plasma membrane domains, respectively, is maintained in cells of ZO-2(-)(/)(-) embryos. However, the architecture of the apical junctional complex is altered, and paracellular permeability of a low-molecular-weight tracer is increased in ZO-2(-/-) embryos. Leaky TJs and, given the association of ZO-2 with connexins and several transcription factors, effects on gap junctions and gene expression, respectively, are likely causes for embryonic lethality. Thus, ZO-2 is required for mouse embryonic development, but ZO-3 is dispensable. This is to our knowledge the first report showing that an individual ZO protein plays a nonredundant and critical role in mammalian development.

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Figures

FIG. 1.

FIG. 1.

Targeting of the ZO-2 and ZO-3 loci and genotyping. (A and B) Targeting strategy. Schematic representations of the genomic locus of ZO-2 (A) and ZO-3 (B) showing exon 2 (with the initiation ATG) and exon 3 (gray boxes). The targeting vectors are designed to disrupt exon 2 by the in-frame insertion of a lacZ gene and a _loxP_-flanked neomycin cassette immediately downstream of the ATG codon. Bars indicate the regions to which probes used for Southern blot analysis hybridize; arrows denote the regions where primers used for genotyping anneal. (C to E) Homologous recombination in ESCs. Southern blot of ScaI-digested genomic DNA of selected ESC clones hybridized with labeled DNA probes hybridizing to genomic DNA (C and D) or the neomycin gene (E), as described in panels A and B, for the identification of homologous recombinants. ScaI fragments of 11.5- and 6.7-kb (ZO-2) or 10.6- and 5.8-kb (ZO-3) corresponding to the WT and targeted mutant alleles, respectively, are detected in targeted (+/−) ESC clones, whereas only the fragment corresponding to the WT allele is present in controls (+/+). (F and G) Genotyping of transgenic mice. Genomic DNA was amplified by PCR using primers designed to distinguish between WT and mutant alleles, as described in panels A and B. Fragments of 580 bp are 679 bp are indicative of the presence of the recombined mutant allele of ZO-2 and ZO-3, respectively, while the 367-bp and 297-bp fragments denote the WT allele. No _ZO-2_−/− mice were detected in newborn litters. (H) Western blot detection of ZO-3 protein. Lysates of intestine and liver from postnatal day 9 ZO-3+/+ and _ZO-3_−/− littermates were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and blotted with antibodies to ZO-3.

FIG. 2.

FIG. 2.

E-cadherin expression and TJ morphology in intestinal epithelial cells of _ZO-3_−/− mice. (A to D) ZO-3 expression and E-cadherin localization. Sections of intestines from ZO-3+/+ or _ZO-3_−/− littermates were stained with antibodies to ZO-3 (A and B) or E-cadherin (C and D) and fluorescently labeled secondary antibodies. (E and F) TJ morphology. Electron micrographs (EM) of intestinal sections of ZO-3+/+ and _ZO-3_−/− mice showing the electron-dense plaques of the apical junctional complex between adjacent epithelial cells. Bars, 0.5 μm (E) and 1 μm (F).

FIG. 3.

FIG. 3.

Postimplantation development of _ZO-2_−/− embryos. Histology of embryos with typical normal or abnormal appearance at E5.5 (A and B), E6.5 (C and D), E7.5 (E and F), and E8.5 (G and H). Note the developmental arrest of _ZO-2_−/− embryos from E5.5 onwards and eventual resorption. epc, extraplacental cone; ac, amniotic cavity, ecc, exocoelomic cavity; epca, ectoplacental cavity; ne, neural ectoderm.

FIG. 4.

FIG. 4.

Developmental arrest of _ZO-2_−/− embryos. ZO-2+/+ and _ZO-2_−/− embryos dissected from their deciduas at E6.5, E7.5, and E8.5. Note the overall smaller size and developmental arrest of _ZO-2_−/− embryos. epc, extraplacental cone.

FIG. 5.

FIG. 5.

Cell proliferation is compromised in E6.5 _ZO-2_−/− embryos. ZO-2+/+ (A and B) and _ZO-2_−/− (C and D) E6.5 embryos were labeled with BrdU and incorporated BrdU (green; A and C), and nuclei (blue; B andD) were visualized in sections by fluorescence microscopy. pac, proamniotic cavity; epc, extraplacental cone.

FIG. 6.

FIG. 6.

Enhanced apoptosis in E7.5 _ZO-2_−/− embryos. Apoptosis in _ZO-2_−/− (A, B, E, and F) and ZO-2+/+ (C and D) embryos at E6.5 (A and B) or E7.5 (C to F) was visualized by fluorescence microscopy using the TUNEL assay (red; A, C, and E). Nuclei are stained in blue (B, D, and F). a, amnion; ac, amniotic cavity; ee, embryonic ectoderm, em, embryonic mesoderm, een, embryonic endoderm.

FIG. 7.

FIG. 7.

_ZO-2_−/− blastocysts differentiate inner cell mass and trophoblast tissue in vitro and express the mesoderm marker T gene. (A to F) Blastocyst cultures. Micrographs of E3.5 ZO-2+/+ (A and D), ZO-2+/− (B and E), or _ZO-2_−/− (C and F) blastocysts after isolation (day 0 [D0]) (A to C) or 4-day (D4) in vitro culture (D to F). Note the normal development of inner cell mass (ICM) and trophoblast outgrowth (TG) for _ZO-2_−/− blastocysts. (G) Genotyping. Blastocysts were genotyped by reverse transcription-PCR, yielding 367-bp and 580-bp fragments for the WT and inactivated ZO-2 alleles, respectively. (H) T-gene expression. Reverse transcription-PCR was used to assess the expression of T gene in ZO-2+/+ or _ZO-2_−/− ESCs and EBs.

FIG. 8.

FIG. 8.

Distribution of ZO-1 and ZO-3 in _ZO-2_−/− embryos is not altered. Sections of E7.5 ZO-2+/+ (A, C, and E) and _ZO-2_−/− (B, D, and F) embryos were labeled with antibodies to ZO-2 (A and B), ZO-1 (C and D), or ZO-3 (E and F) and visualized by fluorescence microscopy. a, amnion; ac, amniotic cavity; ee, embryonic ectoderm; een, embryonic endoderm; exen, extraembryonic endoderm; exe, extraembryonic ectoderm.

FIG. 9.

FIG. 9.

Apical-basal polarity is not affected in _ZO-2_−/− embryos. Sections of E7.5 ZO-2+/+ (A and C) and _ZO-2_−/− (B and D) embryos were labeled with antibodies to the apical marker prominin (A and B) and the lateral maker E-cadherin (B and D) and visualized by fluorescence microscopy. ac, amniotic cavity; ee, embryonic ectoderm; een, extraembryonic endoderm; ee, embryonic ectoderm; embryonic mesoderm.

FIG. 10.

FIG. 10.

The structure and permeability barrier of the apical junctional complex are altered in cells of _ZO-2_−/− embryos. (A to D) Structure. E6.5 (data not shown) and E7.5 embryo sections were analyzed by transmission electron microscopy to visualize the apical junctional complex. Typical electron-dense plaques were readily detected at the apical pole of the lateral membrane of adjacent cells of control embryos (A and C) but were rarely observed in cells of _ZO-2_−/− embryos (B and D). _ZO-2_−/− embryos were identified based on their small size compared to WT and heterozygous embryos. Bars, 5 μm (A and B) and 0.5 μm (C and D). (E and F) Lanthanum permeability. E7.5 embryos were postfixed in lanthanum nitrate and processed for transmission electron microscopy. Note the presence of lanthanum (black arrows) in intercellular spaces of _ZO-2_−/− (E) but not control (F) embryos. Bar, 2 μm. (G and H) Higher magnification of the TJs highlighted with square brackets in panels E and F. Bar, 1 μm.

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