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. 2008 Jun;19(6):2465-75.
doi: 10.1091/mbc.e07-12-1215. Epub 2008 Mar 19.

Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells

Tatsuya Katsuno  1 , Kazuaki Umeda, Takeshi Matsui, Masaki Hata, Atsushi Tamura, Masahiko Itoh, Kosei Takeuchi, Toshihiko Fujimori, Yo-ichi Nabeshima, Tetsuo Noda, Shoichiro Tsukita, Sachiko Tsukita
Affiliations

Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells

Tatsuya Katsuno et al. Mol Biol Cell. 2008 Jun.

Abstract

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1(-/-)) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1(-/-) embryos, and no viable Tjp1(-/-) embryos were observed beyond E11.5. Tjp1(-/-) embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1(+/+) yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1(-/-) yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development.

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Figures

Figure 1.
Figure 1.
Generation of ZO-1–deficient mice. (A) Restriction maps of the wild type allele, the targeting vector, and the targeted allele of the ZO-1 gene. The targeting vector contained the pgk-neo cassette in its middle portion to delete the two to three exons in the targeted allele. The position of the probe for Southern blotting is indicated as a bar (Probe). Ev, EcoRV; Ps, PstI; EI, EcoRI; B, BsrDI; and Pv, PvuII. (B) Genotypic analysis by Southern blotting of EcoRI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice for the ZO-1 gene allele. Southern blotting with the probe indicated in A yields 21- and 8-kb bands from the wild-type and targeted allele, respectively. (C) Loss of ZO-1 protein in the embryonic extract of Tjp1−/− mice examined by immunoblotting. Anti-ZO-1 immunoblotting of the embryonic protein extracts of ZO-1–deficient mice. Embryonic protein extracts (10 μg) from Tjp1+/+ (+/+), Tjp1+/− (+/−), and Tjp1−/− (−/−) mice were immunoblotted with anti-ZO-1 mAb. In the wild-type and heterozygous embryonic extracts, the ZO-1 band is detected, whereas in the homozygous extract, this band is not detected.
Figure 2.
Figure 2.
Macroscopic analysis of Tjp1+/+ and Tjp1−/− embryos at E8.5-E10.5. (A) Photographs showing freshly dissected Tjp1+/+ and Tjp1−/− embryos. Slight growth retardation was obvious in Tjp1−/− embryos compared with Tjp1+/+ embryos at E8.5 (E8.5). From E9.5 onward, Tjp1−/− embryos were markedly smaller than Tjp1+/+ embryos, and almost all failed to turn, a process that occurs in Tjp1+/+ embryos around E9.0 (E9.5). Despite the obvious presence of an allantois (arrowhead) at E9.5 and E10.5, chorioallantoic fusion did not occur in the Tjp1−/− embryos. During E8.5 to E10.5, the Tjp1−/− embryo proper was approximately the same size, although the yolk sac was enlarged to a normal size, compared with Tjp1+/+ litter embryos. The large vitelline vessels (arrows) were detected in Tjp1+/+ yolk sac at E9.5 and E10.5. (B) PECAM-1 stained embryo proper. Although the growth was critically retarded, vasculogenesis, and angiogenesis occurred in the Tjp1+/+ and Tjp1−/− embryo proper. (C) PECAM-1–stained embryonic yolk sac. Numerous large, branching vessels were present in Tjp1+/+ yolk sacs as revealed by PECAM-1 staining, showing the process of angiogenesis. In contrast, the arrangement of branching vessels was not detected extraembryonically in Tjp1−/− yolk sacs, without signs of angiogenesis. Bars, 1 mm.
Figure 3.
Figure 3.
Histological analysis of transverse sections of Tjp1+/+ and Tjp1−/− embryos at E9.0. (A) Caudal regions. The posterior of Tjp1+/+ and Tjp1−/− embryos proper showed a normal neural tube (nt), dorsal aorta (da), and hindgut (hg), and an abnormal morphology of notochord (nc) in Tjp1−/− embryos. (B) Primitive heart. No obvious change was discerned between Tjp1+/+ and Tjp1−/− embryos. (C) Allantois. Although Tjp1−/− allantois was associated with apoptosis, a well-defined vascular network was developed similar to the Tjp1+/+ allantois. (D) Placentas. A transverse sectional analysis of Tjp1+/+ placentas showed the presence of immature embryonic erythrocytes (arrows) and unnucleated maternal erythrocytes (arrowheads), whereas the labyrinth layer of Tjp1−/− embryos lacked immature embryonic nucleated erythrocytes and embryonic blood vessels derived from the mesoderm. Left bottom, high-magnification images of the boxed regions. (E) Yolk sac. In the Tjp1+/+ yolk sac, the vessels were well formed, but in the Tjp1−/− yolk sac, the vessels were dramatically enlarged. Specific structures are as follows: en, extraembryonic endoderm; me, extraembryonic mesoderm; i.e., immature erythrocyte; and ec, endothelial cell. Bars, 100 μm.
Figure 4.
Figure 4.
Apoptosis in Tjp1+/+ and Tjp1−/− embryos at E8.5 to E9.5. (A) Hematoxylin- and eosin-stained transverse sectional images of embryos at E9.0 around the notochord. In Tjp1−/− embryos, the border of the notochord (nc) seemed to be disorganized. Tjp1−/− embryos showed the presence of apoptotic or necrotic cells (arrows) at the notochord, neural tube, hindgut, and mesenchym around them. Specific structures are as follows: nt, neural tube; hg, hind gut; and nc, notochord. Bar, 20 μm. (B) Immunofluorescence images for Ki-67, caspase-3, and DAPI. At E8.5, almost no changes were detected between Tjp1+/+ and Tjp1−/− embryos. At E9.5, in Tjp1−/− embryos, caspase-3–positive apoptotic cells scattered beyond the normal bordered layer of the notochord or neural tube, whereas little apoptotic cells were detected in litter Tjp1+/+ embryos. (C) Quantification of Ki-67 and caspase-3–positive cells (mean and SE [n = 6]). Caspase-3 signals suggested that in Tjp1−/− embryos, the apoptotic cells were significantly increased compared with Tjp1+/+ embryos. Bars, 50 μm.
Figure 5.
Figure 5.
Immunolabeling of ZO-1, ZO-2, and ZO-3 in E8.5 wild-type Tjp1+/+ embryos. (A) a–d, caudal parts of embryos. a′-d′, high magnification of the boxed region of caudal parts of embryos, each corresponding to a–d, respectively. ZO-1 and ZO-2 localized at cell–cell junctions in the mesenchymal cells (arrows). e–h, cephalic parts of embryos. i–l, primitive heart. m–p, yolk sac. q–t, allantois. Specific. structures are as follows: ng, neural groove; fg, foregut; nt, neural tube; so, somite; en, extraembryonic endoderm; and me, extraembryonic mesoderm. Bars, 50 μm (a–d and e–t), 20 μm (a′–d′). (B) Immunofluorescence images of frozen sections of Tjp1+/+ and Tjp1−/− yolk sacs at E9.5 stained for ZO-1, ZO-2, and ZO-3, respectively. In the wild-type Tjp1+/+ yolk sac, ZO-1 was expressed in the extraembryonic endoderm and mesoderm, whereas in the homozygous Tjp1−/− yolk sac, signals for ZO-1 became undetectable. ZO-2 and ZO-3 were expressed only in extraembryonic endoderm in Tjp1+/+ embryos. Note that the ZO-2 signal was apically concentrated in the Tjp1−/− extraembryonic endoderm. Specific structures are as follows: en, extraembryonic endoderm; and me, extraembryonic mesoderm. Bars, 50 μm.
Figure 6.
Figure 6.
Analysis of yolk sac extraembryonic vascular development. (A) Immunolabeling for PECAM-1 in whole-mount yolk sacs of E8.5 and E9.5 embryos of Tjp1+/+ and Tjp1−/− mice. The immunostaining revealed whole patterns of vasculate not differing between Tjp1+/+ and Tjp1−/− embryos at E8.5, although at E9.5, PECAM-1–stained vasculates were quite different. Tjp1−/− extraembryonic mesoderm lacked the fine branched arrangements of vessels constituted by angiogenesis. The connection sites between extraembryonic endoderm and mesoderm were indicated by arrowheads. (B) High-resolution immunofluorescence micrographs labeled for PECAM-1, VE-cadherin, and JAM-A. It was noted that the immunofluorescently labeled patterns for JAM-A, but not for PECAM-1 and VE-cadherin, differed between E8.5 Tjp1+/+ and Tjp1−/− yolk sac. Bars, 100 μm (A) and 25 μm (B).
Figure 7.
Figure 7.
Paracellular barrier assay of the Tjp1+/+ and Tjp1−/− yolk sac at E9.5. (A) Biotinylation from the outside of yolk sacs in Tjp1+/+ and Tjp1−/− embryos. Conjugated biotin was detected with avidin-Texas Red, which did not penetrate the paracellular barrier. (B) Biotinylation from the inside of yolk sac in Tjp1+/+ and Tjp1−/− embryos. When biotin was subcutaneously injected into the excoelomic cavity (*), it was spread beyond the extraembryonic mesodermal cell sheets, showing no barrier function in the extraembryonic mesoderm. Bar, 50 μm.
Figure 8.
Figure 8.
Whole-mount immunofluorescence micrographs of extraembryonic endoderm and mesoderm for cell–cell adhesion-related proteins in Tjp1+/+ and Tjp1−/− mice at E 9.5. In Tjp1+/+ and Tjp1−/− extraembryonic endoderm, the immunofluorescence patterns for various types of cell adhesion-related proteins revealed the normal formation of the epithelial cell sheets with TJs. It is noteworthy that the ZO-2 signal was concentrated in cell–cell adhesion sites of the Tjp1−/− extraembryonic endoderm. Furthermore, the immunofluorescence patterns of TJ-related proteins such as occludin, claudin-6, and JAM-A were also concentrated in cell–cell adhesion sites of the Tjp1−/− extraembryonic endoderm. In contrast, in extraembryonic mesoderm, the immunofluorescence patterns of afadin and α/β-catenin substantiated cell sheet formation but without concentration of TJ-proper proteins, such as occludin, claudin, and JAM-A. Note that in ZO-1−/− extraembryonic mesoderm, the signals for ZO-1/2/3 were not detected. Bar, 50 μm.
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