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Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, Togawa A, Takahashi K, Nishioka H, Yoshida H, Mizoguchi A, Nishikawa S, Takai Y - J. Cell Biol. (1999)

Bottom Line: Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm.Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies.These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka 565-0871, Japan.

ABSTRACT
Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

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Expression and localization of l-afadin during early embryogenesis. Embryos at various stages were subjected to immunofluorescence microscopy and/or whole-mount immunohistochemistry. (A) Immunofluorescence microscopy of E6.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Aa) F-actin; (Ab) l-afadin. (B) Whole-mount immunohistochemistry of E7.0 embryos (lateral view). The samples were stained with the polyclonal anti–l-afadin antibody. (C) Immunofluorescence microscopy of E7.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Ca, Cb, and Cd) F-actin; (Cc and Ce) l-afadin; (Cb and Cc) double staining image with a high magnification of the indicated box b,c in Ca; and (Cd and Ce) double staining image with a high magnification of the indicated box d,e in Ca. (D) Whole-mount immunohistochemistry and immunofluorescence microscopy of E8.5 embryos. The samples were stained with the polyclonal anti–l-afadin antibody for whole-mount immunohistochemistry or with rhodamine-phalloidin and the anti–l-afadin antibody for immunofluorescence microscopy. (Da) Whole-mount immunohistochemistry (ventral view); (Db–Df) immunofluorescence microscopy (transverse section); (Db, Dc, and De) F-actin; (Dd and Df) l-afadin; (Dc and Dd) double staining image with a high magnification of the indicated box c,d in Db; and (De and Df) double staining image with a high magnification of the indicated box e,f in Db. Arrows indicate junctional complex regions. Asterisks indicate l-afadin–negative ectoderm. ee, embryonic ectoderm; ve, visceral endoderm; ps, primitive streak; ms, mesoderm; nf, neural fold; ac, amniotic cavity; ne, neuroepithelium; nt, neural tube; pm, paraxial mesoderm; tb, tail bud; ppc, pericardio-peritoneal canal; s, somite; ic, intraembryonic coelomic cavity. Bars, 30 μm (Aa, Ab, Cb–Ce, and Dc–Df); 100 μm (B, Ca, and Db); and 150 μm (Da).
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Figure 1: Expression and localization of l-afadin during early embryogenesis. Embryos at various stages were subjected to immunofluorescence microscopy and/or whole-mount immunohistochemistry. (A) Immunofluorescence microscopy of E6.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Aa) F-actin; (Ab) l-afadin. (B) Whole-mount immunohistochemistry of E7.0 embryos (lateral view). The samples were stained with the polyclonal anti–l-afadin antibody. (C) Immunofluorescence microscopy of E7.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Ca, Cb, and Cd) F-actin; (Cc and Ce) l-afadin; (Cb and Cc) double staining image with a high magnification of the indicated box b,c in Ca; and (Cd and Ce) double staining image with a high magnification of the indicated box d,e in Ca. (D) Whole-mount immunohistochemistry and immunofluorescence microscopy of E8.5 embryos. The samples were stained with the polyclonal anti–l-afadin antibody for whole-mount immunohistochemistry or with rhodamine-phalloidin and the anti–l-afadin antibody for immunofluorescence microscopy. (Da) Whole-mount immunohistochemistry (ventral view); (Db–Df) immunofluorescence microscopy (transverse section); (Db, Dc, and De) F-actin; (Dd and Df) l-afadin; (Dc and Dd) double staining image with a high magnification of the indicated box c,d in Db; and (De and Df) double staining image with a high magnification of the indicated box e,f in Db. Arrows indicate junctional complex regions. Asterisks indicate l-afadin–negative ectoderm. ee, embryonic ectoderm; ve, visceral endoderm; ps, primitive streak; ms, mesoderm; nf, neural fold; ac, amniotic cavity; ne, neuroepithelium; nt, neural tube; pm, paraxial mesoderm; tb, tail bud; ppc, pericardio-peritoneal canal; s, somite; ic, intraembryonic coelomic cavity. Bars, 30 μm (Aa, Ab, Cb–Ce, and Dc–Df); 100 μm (B, Ca, and Db); and 150 μm (Da).

Mentions: We first examined expression and localization of l-afadin during mouse early embryogenesis. At embryonic day 6.5 (E6.5), embryos developed to egg cylinders containing embryonic and extraembryonic regions and proamniotic cavities. The embryonic ectoderm was composed of high columnar epithelial cells surrounded by the visceral endoderm. Yolk sac and ectoplacental cone were clearly observed in this stage. Immunofluorescence microscopy of E6.5 embryos revealed that l-afadin was localized at the most apical regions of cell–cell adhesion sites, called the junctional complex regions, of the entire embryonic ectoderm, whereas the signals of F-actin were observed along the entire cell surface (Fig. 1 Aa and Ab). l-Afadin was hardly detected in the extraembryonic regions such as the visceral endoderm. At E7.0, embryos underwent primitive streak formation and mesoderm generation. Gastrulation began by the recruitment of embryonic ectodermal cells to the primitive streak, followed by exfoliation of cells from the primitive streak. Whole-mount immunohistochemistry revealed marked expression of l-afadin in the primitive streak and the migrating paraxial mesoderm (Fig. 1 B). At E7.5, l-afadin was highly concentrated at the junctional complex regions in the primitive streak region (neuroepithelium) and the neural fold/groove region, but it was hardly detected in other areas of the ectoderm (Fig. 1, Ca–Ce). It remains to be clarified whether l-afadin is downregulated or whether the proportion of ectodermal cells with low expression of l-afadin increases. By E8.5, normal embryos completed gastrulation and began organogenesis. The primitive streak regressed and newly organized tissues developed. High expression of l-afadin was detected in the tail bud, somites, and the paraxial mesoderm, which is being reorganized to form somites, neural tube, and intraembryonic coelomic cavity/pericardio-peritoneal canal that gives rise to pleura and pericardium (Fig. 1 Da). l-Afadin was highly concentrated at the junctional complex regions in neural tube, somites, and pericardio-peritoneal canal (Fig. 1, Db–Df). These results indicate that l-afadin is highly expressed in a restricted set of epithelial structures and highly concentrated at their junctional complex regions.


Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, Togawa A, Takahashi K, Nishioka H, Yoshida H, Mizoguchi A, Nishikawa S, Takai Y - J. Cell Biol. (1999)

Expression and localization of l-afadin during early embryogenesis. Embryos at various stages were subjected to immunofluorescence microscopy and/or whole-mount immunohistochemistry. (A) Immunofluorescence microscopy of E6.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Aa) F-actin; (Ab) l-afadin. (B) Whole-mount immunohistochemistry of E7.0 embryos (lateral view). The samples were stained with the polyclonal anti–l-afadin antibody. (C) Immunofluorescence microscopy of E7.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Ca, Cb, and Cd) F-actin; (Cc and Ce) l-afadin; (Cb and Cc) double staining image with a high magnification of the indicated box b,c in Ca; and (Cd and Ce) double staining image with a high magnification of the indicated box d,e in Ca. (D) Whole-mount immunohistochemistry and immunofluorescence microscopy of E8.5 embryos. The samples were stained with the polyclonal anti–l-afadin antibody for whole-mount immunohistochemistry or with rhodamine-phalloidin and the anti–l-afadin antibody for immunofluorescence microscopy. (Da) Whole-mount immunohistochemistry (ventral view); (Db–Df) immunofluorescence microscopy (transverse section); (Db, Dc, and De) F-actin; (Dd and Df) l-afadin; (Dc and Dd) double staining image with a high magnification of the indicated box c,d in Db; and (De and Df) double staining image with a high magnification of the indicated box e,f in Db. Arrows indicate junctional complex regions. Asterisks indicate l-afadin–negative ectoderm. ee, embryonic ectoderm; ve, visceral endoderm; ps, primitive streak; ms, mesoderm; nf, neural fold; ac, amniotic cavity; ne, neuroepithelium; nt, neural tube; pm, paraxial mesoderm; tb, tail bud; ppc, pericardio-peritoneal canal; s, somite; ic, intraembryonic coelomic cavity. Bars, 30 μm (Aa, Ab, Cb–Ce, and Dc–Df); 100 μm (B, Ca, and Db); and 150 μm (Da).
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Related In: Results  -  Collection

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Figure 1: Expression and localization of l-afadin during early embryogenesis. Embryos at various stages were subjected to immunofluorescence microscopy and/or whole-mount immunohistochemistry. (A) Immunofluorescence microscopy of E6.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Aa) F-actin; (Ab) l-afadin. (B) Whole-mount immunohistochemistry of E7.0 embryos (lateral view). The samples were stained with the polyclonal anti–l-afadin antibody. (C) Immunofluorescence microscopy of E7.5 embryos (transverse section). The samples were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody. (Ca, Cb, and Cd) F-actin; (Cc and Ce) l-afadin; (Cb and Cc) double staining image with a high magnification of the indicated box b,c in Ca; and (Cd and Ce) double staining image with a high magnification of the indicated box d,e in Ca. (D) Whole-mount immunohistochemistry and immunofluorescence microscopy of E8.5 embryos. The samples were stained with the polyclonal anti–l-afadin antibody for whole-mount immunohistochemistry or with rhodamine-phalloidin and the anti–l-afadin antibody for immunofluorescence microscopy. (Da) Whole-mount immunohistochemistry (ventral view); (Db–Df) immunofluorescence microscopy (transverse section); (Db, Dc, and De) F-actin; (Dd and Df) l-afadin; (Dc and Dd) double staining image with a high magnification of the indicated box c,d in Db; and (De and Df) double staining image with a high magnification of the indicated box e,f in Db. Arrows indicate junctional complex regions. Asterisks indicate l-afadin–negative ectoderm. ee, embryonic ectoderm; ve, visceral endoderm; ps, primitive streak; ms, mesoderm; nf, neural fold; ac, amniotic cavity; ne, neuroepithelium; nt, neural tube; pm, paraxial mesoderm; tb, tail bud; ppc, pericardio-peritoneal canal; s, somite; ic, intraembryonic coelomic cavity. Bars, 30 μm (Aa, Ab, Cb–Ce, and Dc–Df); 100 μm (B, Ca, and Db); and 150 μm (Da).
Mentions: We first examined expression and localization of l-afadin during mouse early embryogenesis. At embryonic day 6.5 (E6.5), embryos developed to egg cylinders containing embryonic and extraembryonic regions and proamniotic cavities. The embryonic ectoderm was composed of high columnar epithelial cells surrounded by the visceral endoderm. Yolk sac and ectoplacental cone were clearly observed in this stage. Immunofluorescence microscopy of E6.5 embryos revealed that l-afadin was localized at the most apical regions of cell–cell adhesion sites, called the junctional complex regions, of the entire embryonic ectoderm, whereas the signals of F-actin were observed along the entire cell surface (Fig. 1 Aa and Ab). l-Afadin was hardly detected in the extraembryonic regions such as the visceral endoderm. At E7.0, embryos underwent primitive streak formation and mesoderm generation. Gastrulation began by the recruitment of embryonic ectodermal cells to the primitive streak, followed by exfoliation of cells from the primitive streak. Whole-mount immunohistochemistry revealed marked expression of l-afadin in the primitive streak and the migrating paraxial mesoderm (Fig. 1 B). At E7.5, l-afadin was highly concentrated at the junctional complex regions in the primitive streak region (neuroepithelium) and the neural fold/groove region, but it was hardly detected in other areas of the ectoderm (Fig. 1, Ca–Ce). It remains to be clarified whether l-afadin is downregulated or whether the proportion of ectodermal cells with low expression of l-afadin increases. By E8.5, normal embryos completed gastrulation and began organogenesis. The primitive streak regressed and newly organized tissues developed. High expression of l-afadin was detected in the tail bud, somites, and the paraxial mesoderm, which is being reorganized to form somites, neural tube, and intraembryonic coelomic cavity/pericardio-peritoneal canal that gives rise to pleura and pericardium (Fig. 1 Da). l-Afadin was highly concentrated at the junctional complex regions in neural tube, somites, and pericardio-peritoneal canal (Fig. 1, Db–Df). These results indicate that l-afadin is highly expressed in a restricted set of epithelial structures and highly concentrated at their junctional complex regions.

Bottom Line: Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm.Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies.These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka 565-0871, Japan.

ABSTRACT
Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

Show MeSH
Related in: MedlinePlus