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Vol. 19, Issue 6, 2465-2475, June 2008
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*Laboratory of Biological Science, Graduate School of Frontier Biosciences, and Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; Department of Biological Science, Nagoya University Graduate School of Science, Nagoya 464-8602, Japan; Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan; KAN Research Institute, Inc., Kobe MI R&D Center, Kobe 650-0047, Japan; Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; and Department of Cell Biology, Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan
Submitted December 6, 2007;
Revised February 25, 2008;
Accepted March 10, 2008
Monitoring Editor: Keith Mostov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Halbleib and Nelson, 2006). In general, cell–cell adhesion molecules are integral membrane proteins that associate with peripheral membrane proteins to regulate and integrate cell–cell adhesion-related phenomena. Zonula occludens (ZO)-1 is a founding member of membrane-associated guanylate kinase (MAGUK) family proteins of tight junctions (TJs), composed of three postsynaptic density 95/disc-large/ZO-1 (PDZ) domains, a Src homology 3 domain, a guanylate kinase (GUK) domain, an acidic domain, and an actin binding region (Itoh et al., 1993; Willott et al., 1993; Jesaitis and Goodenough, 1996; Haskins et al., 1998). It was first defined as an antigen for monoclonal antibodies that recognized TJs in epithelial cells, and it was subsequently revealed as a peripheral membrane protein with a molecular mass of 220 kDa, which underlied the cytoplasmic surface of plasma membranes of TJs (Stevenson et al., 1986). Evidence has accumulated that in epithelial cells, ZO-1 is exclusively located at TJs, but when TJs are not formed, it is concentrated in adherens junctions (AJs) (Itoh et al., 1993, 1999; Furuse et al., 1994; Fanning et al., 1998; Bazzoni et al., 2000). In nonepithelial cells without TJs such as cardiac muscle cells and fibroblasts, ZO-1 is colocalized with cadherins to form AJs (Itoh et al., 1999). ZO-2 was identified as a 160-kDa protein that was coimmunoprecipitated with ZO-1 from cell lysates (Gumbiner et al., 1999). A phosphorylated 130-kDa protein that coimmunoprecipitated with ZO-1 was ZO-3 (Balda et al., 1993). ZO-2 behaved similarly to ZO-1, although with slight functional differences, whereas ZO-3 was much more distinct from ZO-1/2, only detected in epithelia (Inoko et al., 2003), and localized to TJs in a ZO-1/2–dependent way (Wittchen et al., 1999). The molecular basis underlying this distribution is reported that ZO-1 binds to AJ-constitutive peripheral membrane proteins, such as 
-catenin, afadin, and actin, via the N-terminal, N-terminal, and C-terminal half-domains of ZO-1, respectively, thus being linked to the adhesion molecules of AJs such as cadherin and nectin (Itoh et al., 1993, 1997; Yamamoto et al., 1997; Fanning et al., 1998). Conversely, ZO-1 directly binds to adhesion molecules of TJs such as claudins and occludin by PDZ-1 and GUK domains of ZO-1, respectively (Mitic and Anderson, 1998; Gonzalez-Mariscal et al., 2000). The functional roles of ZO-1 in AJs and TJs are supposed to be coordinated by a proper switching mechanism, as a junctional organizer.
In the ZO-1-knockout (KO)/ZO-2-knockdown (KD) epithelial Eph4 cells, in addition to the formation of liner epithelial-typed AJs (zonula adherens [ZA]) being impeded, the formation of claudin-based linear TJs (zonula occludens) was abolished (Umeda et al., 2006). It was recently revealed that when epithelial ZA was formed from primordial AJs, ZO-1/2 regulated the time course of the formation of ZA in a rac-dependent way (Ikenouchi et al., 2007). Although ZO-3 supposedly played a role distinct from that of ZO-1/2, ZO-3 deficiency produced no phenotypes in cells and mice (Adachi et al., 2006). Thus, ZO-1 and its relative ZO-2 are among the factors that play critical roles in the formation and maintenance of ZA and ZO of the cell–cell adhesion apparatus (Umeda et al., 2004, 2006; Hernandez et al., 2007; Ikenouchi et al., 2007).
In this study, we generated ZO-1–deficient mice to explore the function of ZO-1 in vivo. ZO-1–deficient mice died at the embryonic (E) stage around day 10.5, with embryonic and extraembryonic defects. As embryonic defects, an absence of turning was noted in almost all embryos in the macroscopic and microscopic views, and the neural tube and notochord areas as well as allantois were found to be disorganized due to apoptosis. As a yolk sac extraembryonic defect, angiogenesis seemed to be impaired in ZO-1–deficient mice, affecting embryonic development. These results demonstrate a critical role for ZO-1 in early embryonic development due to both of embryonic and extraembryonic effects, suggesting the important role of cell–cell adhesive junctions in tissue organization and remodeling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), rabbit anti-ZO-3 polyclonal antibody (pAb) (Inoko et al., 2003), rat anti-occludin mAb (Saitou et al., 1997), and rabbit anti-junctional adhesion molecule (JAM)-A pAb (Komiya et al., 2005), rabbit anti-claudin-6 pAb (Morita et al., 1999) and rat anti-cingulin mAb (Ohnishi et al., 2004) were used as described previously. Rat anti-mouse E-cadherin mAb (ECCD2) was generously provided by Dr. M. Takeichi (Center for Developmental Biology, Kobe, Japan). Rabbit anti-afadin pAb was generated using amino acids 1447–1822 of afadin. Antibodies for goat anti-ZO-2 and anti-VE-cadherin pAb (Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-platelet/endothelial cell adhesion molecule (PECAM)-1 mAb (BD Biosciences PharMingen, San Diego, CA), rabbit anti-caspase-3 pAb (Cell Signaling Technology, Danvers, MA), rabbit anti--catenin and anti-β-catenin pAb (Sigma Chemical, Poole, Dorset, United Kingdom), and rabbit anti-myosin-2B (MHC-B) pAb (Covance Research Products, Princeton, NJ), as well as rat anti-Ki-67 pAb (Dako Denmark A/S, Glostrup, Denmark), rhodamine-phalloidin (Cytoskeleton, Denver, CO), and 4,6-diamidino-2-phenylindole (DAPI) (Nakarai Tesque, Kyoto, Japan) were purchased commercially.
Generation of ZO-1 Knockout Mice
The 
phage 129/Sv mouse genomic library was screened using mouse ZO-1 cDNA fragments as a probe. For the gene targeting of embryonic stem (ES) cells, a 5.1-kb PstI–BsrDI fragment and a 1.7-kb PvuII fragment were ligated to the targeting vector cassette. The targeting vector containing the β-geo was linearized at a unique SacII site located at the 5' end of the 5' homologous fragment, and then 4 x 107 ES cells were electroporated with 100 µg of linearized targeting vector DNA using a Gene Pulser (Bio-Rad, Hercules, CA) set at 400 V and 25 µF. Cells were plated on feeder cells in DMEM supplemented with 20% fetal calf serum for 48 h, followed by selection with 100 µg/ml Geneticin (G-418). After 8 d, the G-418–resistant colonies were picked up and screened by Southern blotting with the 3' external probe. Correctly targeted clones were identified by additional 8.6-kb band together with the 21-kb band of the wild-type allele. Tail biopsy or embryonic DNA was routinely genotyped by polymerase chain reaction (PCR) by using 30 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 40 s, with the following primers: primer-1, 5'-GTCCACTTAGATCTGGTCTGTCTG-3'; primer-2, 5'-TAGAAACTCACCCTGTGAAGCGTC-3'; and primer-3, 5'-CAAACGGCGGATTGACCGTAATGG-3'.
Dissection of Embryos and Genotyping
Heterozygous mice were bred to obtain wild-type (Tjp1+/+), heterozygote (Tjp1+/–), and homozygous mutant (Tjp1–/–) embryos. Mice were kept on a 12-h light-dark cycle, and the morning of the day on which a vaginal plug was detected was designated E0.5. Embryos were dissected from the uterus in phosphate-buffered saline (PBS), and dissected Reichert's membrane was used for genotyping. Reichert's membrane was digested for 6 h at 60°C in TP lysis buffer (50 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 0.5% SDS, and 5 mM EDTA), containing 200 µg/ml proteinase K (Promega, Annandale, Australia) and boiled for 5 min before being subjected to PCR (as described above).
Immunoblotting
Protein was isolated from whole embryos in lysis buffer (62.5 mM Tris-Cl, pH 6.8, 2% glycerol, 1% SDS, 5 mM EDTA, and a protease inhibitor cocktail [Nakarai Tesque, Kyoto, Japan]), sonicated on ice five times for 3 s, and centrifuged at 48,000 x g for 20 min at room temperature (RT). The supernatant (crude extract) was used for immunoblotting. Proteins separated by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels were electrophoretically transferred onto polyvinylidene difluoride membranes that were then incubated with the primary antibody. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G and the appropriate substrate as described by the manufacturer (GE Healthcare, Chalfont St. Giles, United Kingdom).
Histological Analysis
Embryos were isolated in PBS and photographed. For histological analyses, the embryos were fixed for 1 h in 4% paraformaldehyde (PFA) at RT, and then they were dehydrated and embedded in paraffin. Embryos were sectioned, and the sections were stained with hematoxylin and eosin.
Immunofluorescence Microscopy
Embryos were washed thoroughly with PBS, fixed for 30 min in PBS containing 4% PFA at RT, placed in a solution of 15% sucrose in PBS for 5 h, embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA, Torrance, CA), and frozen using liquid nitrogen. Frozen sections, 6 µm in thickness, were cut on a cryostat, mounted on glass slides, air-dried, washed with PBS three times, and treated with 0.1% Triton X-100/PBS for 10 min. They were then processed for immunofluorescence microscopy as described previously (Adachi et al., 2006). Whole-mount embryos were immunostained for PECAM-1 to detect the vascular endothelium as described previously (Dominguez et al., 2007). For the analysis of immunofluorescence-labeled yolk sac whole mounts, yolk sacs were removed from the embryo after the fixing process, and they were stained as described previously (Umeda et al., 2004, 2006), and then they were attached to glass slides.
Isolation of RNA and Reverse Transcription (RT)-PCR
Total RNA was isolated from Tjp1+/+ and Tjp1–/– yolk sac in E9.5 embryos, using an RNeasy microkit (QIAGEN, Valencia, CA). First-strand cDNAs were generated, using SuperScript II reverse transcriptase, according to the manufacturer's directions (Invitrogen, Carlsbad, CA). The first-strand cDNAs (25 ng) were amplified by PCR using specific nucleotide primers. The primers for Tie-1 and Flt-1 were described previously (Baumer et al., 2006). The primers for PECAM-1, Flk-1, VE-cadherin, Tie-2, βH, and β-actin were also described previously (Gory-Faure et al., 1999). The primers for JAM-A were described previously (Cooke et al., 2006). The primers for ZO-1, ZO-2, and ZO-3 were with the following primers: ZO-1 primer-1, 5'-GCTAAGAGCACAGCAATG GA-3'; ZO-1 primer-2, 5'-GCATGTTCAACGTTATCCAT-3'; ZO-2 primer-1, 5'-CATGGGCGCGGACTATCT-3'; ZO-2 primer-2, 5'-CTGTGGCGGGGAGGTTTGA-3'; ZO-3 primer-1, 5'-CACGCAATCCTGGATGTCA-3'; and ZO-3 primer-2, 5'-GTCGCGCCTGCTGTTGCTGTA-3'. The amplification was performed using a step-down PCR as follows: three cycles: 94°C for 30 s, 69°C for 30 s, 72°C for 40 s; three cycles: 94°C for 30 s, 66°C for 30 s, 72°C for 40 s; three cycles: 94°C for 30 s, 63°C for 30 s, 72°C for 40 s; three cycles: 94°C for 30 s, 60°C for 30 s, 72°C for 40 s; and 25 cycles: 94°C for 30 s, 57°C for 30 s, 72°C for 40 s.
Barrier Assay
The biotin tracer assay was performed using the cell surface biotinylation method as described previously (Umeda et al., 2006), with some modifications. After the decidua and Reichert's membrane were carefully removed, embryos were washed with HEPES-buffered saline (HBS; 25 mM HEPES-NaOH, pH 7.2, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose, and 1.8 mM CaCl2). The endoderm of yolk sac was biotinylated by submerging the conceptus in HBS supplemented with 1 mg/ml EZ-Link Sulfo-NHS-LC-biotin (Pierce Chemical, Rockford, IL). The mesoderm of yolk sac and the embryo proper were biotinylated by injecting the same solution into the exocoelomic cavity using a mouth-held microcapillary pipette (Zeigler et al., 2006). After a 10-min incubation, embryos were washed with HBS, fixed with 4% formaldehyde in HBS for 10 min at RT, and processed for fluorescence microscopy with streptavidin-Texas Red (Calbiochem, San Diego, CA.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 wk, and they showed no obvious phenotypes, with comparable fertility and growth rates to those of wild-type Tjp1+/+ mice (data not shown). Intercrossing Tjp1+/– mice produced offspring, of which 36% were wild-type Tjp1+/+ mice, 64% were heterozygous Tjp1+/– mice, and none were homozygous Tjp1–/– mice, thus leading us to examine the embryos. The examination of >200 embryos at various stages of gestation from ZO-1 heterozygous intercrosses revealed that Tjp1–/– embryos were indistinguishable from wild-type embryos up to E8.5. At E8.5, Tjp1–/– embryos were retarded, lagging at least 0.5 d behind the development of Tjp1+/+ and Tjp1+/– littermates (Figure 2A). By E9.5 and E10.5, Tjp1–/– embryo proper displayed severe growth defects, most apparently, a significant reduction in size and an absence of turning (Figure 2, A and B). Furthermore, chorioallantoic fusion did not occur in the Tjp1–/– embryos (Figure 2, A and B). Defects were also apparent in the Tjp1–/– yolk sac extraembryonic region. Yolk sacs were not fully developed without normal patterning of vascularization in Tjp1–/– embryos, compared with wild-type Tjp1+/+ litter embryos (Figure 2C). Together, defects in Tjp1–/– embryonic tissue were detected in embryonic and extraembryonic regions.
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4-integrin, induced defects in the morphogenesis of the heart (Yang et al., 1995; Radice et al., 1997; Kruger et al., 2000), no obvious change was observed in the primitive heart between Tjp1+/+ and Tjp1–/– embryos (Figure 3B), with a spontaneous beating in the heart until E10.5.
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Defects in Angiogenesis in Tjp1–/– Embryos
The preformed primitive vascular plexus begins to be remodeled in the yolk sacs of Tjp1+/+ embryos by E8.5, so that the yolk sacs have an organized vascular network of branching vessels at E9.5, which are lined with PECAM-1–positive endothelial cells. Although the initial primitive vascular plexus was formed in Tjp1–/– yolk sacs at E8.5, PECAM-1 immunostaining confirmed the lack of any identifiable mature or remodeled blood vessels in Tjp1–/– yolk sacs around E9.5 (Figure 6A). In contrast to the compartmentalization that was developed in Tjp1+/+ embryos, these Tjp1–/– vessels continued to expand without compartments, except for very few remaining adhesion sites, as shown in the cross-sectional images. Even with these severe morphological defects, the endothelial cell layers themselves seem normal in the extraembryonic endoderm and mesoderm in Tjp1–/– embryos (Figure 3E). When the expression of endothelial markers and hematopoietic markers was analyzed by RT-PCR using E9.5 Tjp1+/+ and Tjp1–/– yolk sacs (Supplemental Figure 2), it was revealed that in Tjp1–/– yolk sacs, all markers were expressed normally, in the same way as those in Tjp1+/+ yolk sacs. In contrast to PECAM-1 and VE-cadherin, which showed the same localization in cell–cell adhesion sites between Tjp1+/+ and Tjp1–/– yolk sacs, JAM-A was localized in the cell–cell adhesion sites of Tjp1+/+ yolk sacs, but it was not localized there in Tjp1–/– yolk sacs (Figure 6B). These results suggested that vasculogenesis occurred normally in Tjp–/– yolk sacs, whereas angiogenesis was abnormal, compared with Tjp1+/+yolk sacs, possibly due to some defects in cell–cell adhesion-related tissue remodeling.
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; Ikenouchi et al., 2007). Because the yolk sac extraembryonic mesoderm lacked ZO-1/2/3 in Tjp1–/– embryos, we examined the morphology of the developing sheets of the yolk sac in detail by whole-mount immunostaining for cell–cell adhesion-related molecules (Figure 8). The immunofluorescence pattern of ZO-1/2/3 in Tjp1+/+ embryos showed a fine linear pattern, typical of TJs, suggesting that these TJ-MAGUK proteins were associated with TJs, although ZO-2 seemed to be more broadly distributed around TJs, not only just on TJs. The clear line stained for the TJ-MAGUK proteins was positive for claudins, occludin, or both, except for yolk sac extraembryonic mesoderm which were positive for ZO-1 but negative for other TJs-related proteins (ZO-2/3, occludin, claudin-6, JAM-A, and cingulin) (Figure 8). In contrast, in the yolk sac Tjp1–/– extraembryonic regions, the immunofluorescence patterns of ZO-2 were slightly changed from a somewhat broad pattern around TJs to a sharper pattern, compared with those of Tjp1+/+ extraembryonic regions, suggesting some functionally redundant role of ZO-2 for ZO-1. In Tjp1–/– yolk sac, the extraembryonic endodermal and mesodermal cell sheet layer was normally formed, suggesting a normal vasculogenesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin binding, with their different molecular weights mainly related to the different number of amino acids in actin binding domains (Mitic and Anderson, 1998; Gonzalez-Mariscal et al., 2000). The cell level analysis showed that a large part of the ZO-1 knockout/ZO-2 knockdown phenotypes were similarly rescued by either ZO-1 or ZO-2; however, in ZO-1-deficient epithelial Eph4 cells, overexpression of ZO-2 does not rescue the delayed formation of TJs (Umeda et al., 2004). In a reasonably consistent way, it was recently reported that ZO-2–deficient Tjp2–/– mice, compared with wild-type embryos, died shortly after implantation because of arrest in early gastrulation with increased apoptosis at E7.5 (Xu et al., 2008). Thus, ZO-1 and ZO-2 were not redundant in the early development of embryos. It is reported that ZO-3 plays a role distinct from that of ZO-1/2 and that ZO-3 deficiency produces no phenotypes at cellular and mouse levels (Umeda et al., 2004, 2006; Adachi et al., 2006; Xu et al., 2008). Hence, the mouse level analysis revealed that the closely related proteins, ZO-1/2/3 were not redundant to each other. In ZO-2–deficient mice, some defects were recognized in the function of the junctional complex, the paracellular barrier function, and also in the tissue organization caused by apoptosis. These might possibly rationalize the earlier timing of lethality of Tjp2–/– mice compared with Tjp1–/– mice, in which defects in the junctional function itself were not detected. The present study showed that the expression patterns of ZO-1 and ZO-2 were basically very similar in wild-type Tjp1+/+ embryos, except in the yolk sac extraembryonic mesoderm in which ZO-1, but not ZO-2 or ZO-3, was expressed. Hence, no ZO-1/2/3 were expressed in the yolk sac extraembryonic mesoderm in Tjp1–/– embryos, in which we could detect the mislocalization of JAM-A, a component of TJs as well as AJs, and defects in angiogenesis compared with Tjp1+/+ mice. Thus, these findings suggested a role for ZO-1/2 in determining cell fate and remodeling in a cell adhesion-related way.
Although the connection of yolk sac mesodermal and endodermal layers, an important angiogenesis process (Drake and Little, 1995; Djonov et al., 2003; Coultas et al., 2005), was significantly inhibited by a deficiency of ZO-1, it remains unclear how the absence of ZO-1 and the mislocalization of JAM-A were related with defective angiogenesis in Tjp1–/– embryos. It is possible that ZO-1, JAM-A, or both played an important role in the remodeling for connection between the yolk sac mesoderm and endoderm layers in angiogenesis by directly or indirectly organizing some aspects of cell–cell adhesion in such a way that its deficiency inhibited angiogenesis. In this respect, it is reported that fibroblast growth factor-2–induced angiogenesis is defective in the JAM-A–deficient mice (Cooke et al., 2006). We could not recognize other abnormal patterns of TJ components in Tjp1–/– mouse epithelia, except for more concentrated ZO-2 staining in junctional patterns compared with Tjp1+/+ epithelia, suggesting a partial redundant role of ZO-2 in place of ZO-1 in Tjp1–/– embryo and extraembryonic regions. However, the embryonic lethal phenotypes of ZO-1- and ZO-2–deficient mice contradicted any redundancy.
In parallel with the possibility that the embryonic hypertrophy was indirectly induced by the defects in extraembryonic angiogenesis, it is also possible that because of some defects related to ZO-1 deficiency in cell remodeling in Tjp1–/– embryos in the notochord, neural tube, hindgut and surrounding mesenchyme, the cells in these regions were directly lead to apoptosis. The restriction of the apoptotic area contrasted to the case of Edd- and connexin-45–deficient mice in which apoptosis occurred almost everywhere in embryos (at E9.5) indirectly caused by extraembryonic defects in angiogenesis. As well, reports on similar phenotypes (Goh et al., 1997; Radice et al., 1997; Kruger et al., 2000; Saunders et al., 2004; Argraves and Drake, 2005; Baumer et al., 2006; Morin-Kensicki et al., 2006; Dominguez et al., 2007) might possibly provide some clues about the mechanistic bases for the phenotypes in Tjp1–/– mice. In the Drosophila tracheal system, the Drosophila homologue of ZO-1, Polychaetoid, is suggested to be involved in AJ remodeling in epithelial morphogenesis, partially consistent with our findings in embryonic and extraembryonic regions (Jung et al., 2006). Thus, it is possible that apoptosis was induced in the specific embryonic region most possibly because of defects in cell modeling in Tjp1–/– mice.
Accumulated cases report that defects in the visceral yolk sac and allantois lead to retarded growth of the embryo and early lethality because of defective blood circulation (Radice et al., 1997; Kruger et al., 2000; Rossant and Cross, 2001; Copp, 1995; Saunders et al., 2004); both of which were defective features in Tjp1–/– embryos. It is noteworthy that the phenotypically related factors were VCAM-1, 4-, 5-integrin, connexin-45, VE-cadherin, and N-cadherin, which were possibly related to cell–cell interactions (Kwee et al., 1995; Yang et al., 1995; Goh et al., 1997; Radice et al., 1997; Gory-Faure et al., 1999; Kruger et al., 2000). In this regard, the secondary effects of extraembryonic defects on embryonic defects could be critically tested by using tetraploid rescue (Mackay and West, 2005) or through the generation of conditional knockout animals in which ZO-1 expression is disrupted only in embryonic tissue. These remain as future issues. As well, a sophisticated assay system seems needed for better functional analysis of ZO-1 in embryogenesis. The establishment of culture systems from Tpj1–/– mice provides a novel way to understand the function of TJ-MAGUK family members and to establish possible therapeutic strategies for related diseases. Studies are presently being conducted along these lines in our laboratory.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present addresses: || Department of Molecular Pharmacology, Graduate School of Medical Science, Kumamoto University, Honjo, Kumamoto 860-8556, Japan; 
¶ Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 135-8550, Japan;
# Department of Pathology, Hyogo College of Medicine, 4-11 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan;
@ Department of Molecular and Cell Biology, Institute of Medical Science, Dokkyo Medical University, Shimotsuga-gun, Tochigi, 321-0293 Japan;
** Department of Anatomy and Developmental Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
Address correspondence to: Sachiko Tsukita (atsukita{at}biosci.med.osaka-u.ac.jp).
Abbreviations used: MAGUK, membrane-associated guanylate kinase; TJ, tight junction; AJ, adherens junction; PDZ, postsynaptic density 95/disc-large/zona occludens; ZA, zonula adherens; ZO, zonula occludens.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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