Deficiency of ZO-1 Causes Embryonic Lethal Phenotype Associated with Defected Yolk Sac Angiogenesis and Apoptosis of Embryonic Cells

Tatsuya Katsuno,* ${dagger}$ Kazuaki Umeda, ${ddagger}$ ${sect}$ || Takeshi Matsui,*¶ Masaki Hata, ${sect}$ # Atsushi Tamura,* Masahiko Itoh, ${ddagger}$ * Kosei Takeuchi, ${dagger}$ ** Toshihiko Fujimori, ${dagger}$ ${dagger}$ Yo-ichi Nabeshima, ${dagger}$ ${dagger}$ Tetsuo Noda, ${ddagger}$ ${ddagger}$ Shoichiro Tsukita, ${ddagger}$ and Sachiko Tsukita*

^*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; Department of Cell Biology, The JFCR-Cancer Institute, Tokyo 135-8550, Japan

Monitoring Editor: Keith Mostov

ZO-1/2/3 are the members ofthe TJ-MAGUK family of membrane-associated guanylate kinasesassociated with tight junctions. To investigate the role ofZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1^-/-) micewere generated by gene targeting. While heterozygous mice showednormal development/fertility, delayed growth/development wereevident from E8.5 onward in Tjp1^-/- embryos and no viable Tjp1^-/-embryos were observed beyond E11.5. Tjp1^-/- embryos exhibitedmassive apoptosis in the notochord, neural tube area and allantoisat E9.5. In the yolk sac, the ZO-1-deficiency induced defectsin vascular development with impaired formation of vasculartrees, along with defective chorioallantoic fusion. Immunostainingof wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2were expressed in almost all embryonic cells, showing tightjunction-localizing patterns, with or without ZO-3 which wasconfined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expressionwithout influence on ZO-2/3-expression. In Tjp1^+/+ yolk sacextraembryonic 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 JAMs. As a result,angiogenesis was defected in Tjp1^-/- yolk sac, though differentiation/positioningof endothelial cells appeared to be normal. In conclusion, ZO-1may affect cell remodeling and defects in tissue organizationin both embryonic and extraembryonic regions, thus playing anessential role for embryonic development.

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)