Limits...
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.

Show MeSH

Related in: MedlinePlus

Disorganized ectoderm in cystic EBs derived from afadin−/− ES cells. Cystic EBs were subjected to histological analysis or immunofluorescence microscopy. For immunofluorescence microscopy, cystic EBs were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody, or with the anti–E-cadherin and anti–ZO-1 antibodies. (A) Cystic EBs derived from wild-type ES cells; (B) cystic EBs derived from afadin−/− ES cells; (Aa and Ba) microscopic appearance; (Ab and Bb) staining with hematoxylin and eosin; (Ac, Ad, Bc, and Bd) double staining with rhodamine-phalloidin and the anti–l-afadin antibody; (Ac and Bc) l-afadin; (Ad and Bd) F-actin; (Ae, Af, Be, and Bf) double staining with the anti–E-cadherin and anti–ZO-1 antibodies; (Ae and Be) E-cadherin; and (Af and Bf) ZO-1. Arrowheads indicate junctional complex regions. en, endodermal layer; cy, cyst cavity; ec, ectodermal layer; rm, Reichert's membranes; and cm, cell mass. Bars, 150 μm (Aa and Ba); 100 μm (Ab and Bb); and 30 μm (Ac–Af and Bc–Bf).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169488&req=5

Figure 8: Disorganized ectoderm in cystic EBs derived from afadin−/− ES cells. Cystic EBs were subjected to histological analysis or immunofluorescence microscopy. For immunofluorescence microscopy, cystic EBs were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody, or with the anti–E-cadherin and anti–ZO-1 antibodies. (A) Cystic EBs derived from wild-type ES cells; (B) cystic EBs derived from afadin−/− ES cells; (Aa and Ba) microscopic appearance; (Ab and Bb) staining with hematoxylin and eosin; (Ac, Ad, Bc, and Bd) double staining with rhodamine-phalloidin and the anti–l-afadin antibody; (Ac and Bc) l-afadin; (Ad and Bd) F-actin; (Ae, Af, Be, and Bf) double staining with the anti–E-cadherin and anti–ZO-1 antibodies; (Ae and Be) E-cadherin; and (Af and Bf) ZO-1. Arrowheads indicate junctional complex regions. en, endodermal layer; cy, cyst cavity; ec, ectodermal layer; rm, Reichert's membranes; and cm, cell mass. Bars, 150 μm (Aa and Ba); 100 μm (Ab and Bb); and 30 μm (Ac–Af and Bc–Bf).

Mentions: When wild-type ES cells were subjected to suspension culture, cells aggregated to form EBs, some of which eventually develop to a two-layered cystic structure consisting of the outer endodermal layer and the inner high columnar ectodermal layer, although EBs with more complex structures with a large yolk sac–like cyst were often observed (Fig. 8, Aa and Ab). During earlier stages of EB formation, afadin−/− ES cells showed no significant difference (data not shown). Moreover, EBs with a large yolk sac–like cyst were often formed, suggesting that the endodermal components function normally (Fig. 8 Ba). On the other hand, in EBs with an amniotic cavity–like cyst, many necrotic cells were found in the cavity, while leaving the outer layer intact (Fig. 8 Bb). Generation of mesodermal cells at the space between the ectodermal and endodermal layers was observed, but the well-organized ectodermal layer did not develop. These results indicate that the ectoderm-specific defects of afadin−/− embryos may be reproduced in the in vitro EB model. Moreover, the presence of cellular components in the EB cavity may reflect the defects in the polarity of the ectodermal layer.


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)

Disorganized ectoderm in cystic EBs derived from afadin−/− ES cells. Cystic EBs were subjected to histological analysis or immunofluorescence microscopy. For immunofluorescence microscopy, cystic EBs were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody, or with the anti–E-cadherin and anti–ZO-1 antibodies. (A) Cystic EBs derived from wild-type ES cells; (B) cystic EBs derived from afadin−/− ES cells; (Aa and Ba) microscopic appearance; (Ab and Bb) staining with hematoxylin and eosin; (Ac, Ad, Bc, and Bd) double staining with rhodamine-phalloidin and the anti–l-afadin antibody; (Ac and Bc) l-afadin; (Ad and Bd) F-actin; (Ae, Af, Be, and Bf) double staining with the anti–E-cadherin and anti–ZO-1 antibodies; (Ae and Be) E-cadherin; and (Af and Bf) ZO-1. Arrowheads indicate junctional complex regions. en, endodermal layer; cy, cyst cavity; ec, ectodermal layer; rm, Reichert's membranes; and cm, cell mass. Bars, 150 μm (Aa and Ba); 100 μm (Ab and Bb); and 30 μm (Ac–Af and Bc–Bf).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169488&req=5

Figure 8: Disorganized ectoderm in cystic EBs derived from afadin−/− ES cells. Cystic EBs were subjected to histological analysis or immunofluorescence microscopy. For immunofluorescence microscopy, cystic EBs were doubly stained with rhodamine-phalloidin and the polyclonal anti–l-afadin antibody, or with the anti–E-cadherin and anti–ZO-1 antibodies. (A) Cystic EBs derived from wild-type ES cells; (B) cystic EBs derived from afadin−/− ES cells; (Aa and Ba) microscopic appearance; (Ab and Bb) staining with hematoxylin and eosin; (Ac, Ad, Bc, and Bd) double staining with rhodamine-phalloidin and the anti–l-afadin antibody; (Ac and Bc) l-afadin; (Ad and Bd) F-actin; (Ae, Af, Be, and Bf) double staining with the anti–E-cadherin and anti–ZO-1 antibodies; (Ae and Be) E-cadherin; and (Af and Bf) ZO-1. Arrowheads indicate junctional complex regions. en, endodermal layer; cy, cyst cavity; ec, ectodermal layer; rm, Reichert's membranes; and cm, cell mass. Bars, 150 μm (Aa and Ba); 100 μm (Ab and Bb); and 30 μm (Ac–Af and Bc–Bf).
Mentions: When wild-type ES cells were subjected to suspension culture, cells aggregated to form EBs, some of which eventually develop to a two-layered cystic structure consisting of the outer endodermal layer and the inner high columnar ectodermal layer, although EBs with more complex structures with a large yolk sac–like cyst were often observed (Fig. 8, Aa and Ab). During earlier stages of EB formation, afadin−/− ES cells showed no significant difference (data not shown). Moreover, EBs with a large yolk sac–like cyst were often formed, suggesting that the endodermal components function normally (Fig. 8 Ba). On the other hand, in EBs with an amniotic cavity–like cyst, many necrotic cells were found in the cavity, while leaving the outer layer intact (Fig. 8 Bb). Generation of mesodermal cells at the space between the ectodermal and endodermal layers was observed, but the well-organized ectodermal layer did not develop. These results indicate that the ectoderm-specific defects of afadin−/− embryos may be reproduced in the in vitro EB model. Moreover, the presence of cellular components in the EB cavity may reflect the defects in the polarity of the ectodermal layer.

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