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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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Developmental defects of afadin−/− embryos during early embryogenesis. (A) Gross appearance. (Aa) E7.5 embryos; (Ab) E8.5 embryos; and (Ac) E9.5 embryos: left, wild-type embryos; and right, afadin−/− embryos. (B) Histological analysis of E7.5 embryos. E7.5 embryos were subjected to staining with hematoxylin and eosin. (Ba) Wild-type embryos (transverse section); (Bb–Be) afadin−/− embryos; (Bb) transverse section of the proximal region; (Bc) transverse section of the middle region; (Bd) transverse section of the distal region; and (Be) sagittal section. (C) Histological analysis of E8.5 embryos. E8.5 embryos were subjected to staining with hematoxylin and eosin. (Ca) Wild-type embryos (transverse section); and (Cb) afadin−/− embryos (transverse section). hf, headfold; ps, primitive streak; ec, ectoplacental cone; ht, primitive heart; ee, embryonic ectoderm; ve, visceral endoderm; ac, amniotic cavity; ms, mesoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm; ecc, exocoelomic cavity; ic, intraembryonic coelomic cavity; ng, neural groove; and s, somite. Bars, 150 μm (Aa, Ba–Be, Ca, and Cb); 300 μm (Ab); and 350 μm (Ac).
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Figure 3: Developmental defects of afadin−/− embryos during early embryogenesis. (A) Gross appearance. (Aa) E7.5 embryos; (Ab) E8.5 embryos; and (Ac) E9.5 embryos: left, wild-type embryos; and right, afadin−/− embryos. (B) Histological analysis of E7.5 embryos. E7.5 embryos were subjected to staining with hematoxylin and eosin. (Ba) Wild-type embryos (transverse section); (Bb–Be) afadin−/− embryos; (Bb) transverse section of the proximal region; (Bc) transverse section of the middle region; (Bd) transverse section of the distal region; and (Be) sagittal section. (C) Histological analysis of E8.5 embryos. E8.5 embryos were subjected to staining with hematoxylin and eosin. (Ca) Wild-type embryos (transverse section); and (Cb) afadin−/− embryos (transverse section). hf, headfold; ps, primitive streak; ec, ectoplacental cone; ht, primitive heart; ee, embryonic ectoderm; ve, visceral endoderm; ac, amniotic cavity; ms, mesoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm; ecc, exocoelomic cavity; ic, intraembryonic coelomic cavity; ng, neural groove; and s, somite. Bars, 150 μm (Aa, Ba–Be, Ca, and Cb); 300 μm (Ab); and 350 μm (Ac).

Mentions: Embryos were isolated at various stages of gestation and their genotypes were determined (Table ). Distribution of each genotype examined at E7.5–E9.5 followed the Mendelian law, whereas no homozygous embryos were detected from E10.5. At E6.5, gross morphological analysis did not distinguish the homozygous embryos from the wild-type and heterozygous littermates (data not shown), indicating that implantation and egg cylinder formation occur normally in the absence of afadin. To examine the presence of residual maternal afadin, which may affect implantation and egg cylinder formation in the homozygous embryos, preimplantation embryos at E3.5 (early blastocysts) were cultured and their levels of l-afadin were determined by immunofluorescence microscopy. Of 20 embryos examined, 16 showed weak but significant staining, whereas the remaining did not show any signal (data not shown). It is most likely that the latter embryos are homozygous and that there is no residual maternal afadin in the homozygous embryos. In contrast to embryos at E6.5, it was easy to distinguish the homozygous embryos from the wild-type and heterozygous littermates in embryos at E7.5–E9.5. Compared with wild-type embryos, the architecture of the homozygous embryos was apparently distorted and reduced in size (Fig. 3, Aa–Ac). Of note, however, is that their extraembryonic regions, including ectoplacental cone and yolk sac, developed normally, indicating that the anomalies are basically restricted to embryos proper. Whereas the anterior–posterior distinction could easily be made by gross appearance, and some vascular system and blood were detectable, the homozygous embryos were always flat and short, and no landmark tissues such as heart developed at this stage. The anomalies specific for these embryos proper in afadin−/− embryos are consistent with the expression patterns of l-afadin.


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)

Developmental defects of afadin−/− embryos during early embryogenesis. (A) Gross appearance. (Aa) E7.5 embryos; (Ab) E8.5 embryos; and (Ac) E9.5 embryos: left, wild-type embryos; and right, afadin−/− embryos. (B) Histological analysis of E7.5 embryos. E7.5 embryos were subjected to staining with hematoxylin and eosin. (Ba) Wild-type embryos (transverse section); (Bb–Be) afadin−/− embryos; (Bb) transverse section of the proximal region; (Bc) transverse section of the middle region; (Bd) transverse section of the distal region; and (Be) sagittal section. (C) Histological analysis of E8.5 embryos. E8.5 embryos were subjected to staining with hematoxylin and eosin. (Ca) Wild-type embryos (transverse section); and (Cb) afadin−/− embryos (transverse section). hf, headfold; ps, primitive streak; ec, ectoplacental cone; ht, primitive heart; ee, embryonic ectoderm; ve, visceral endoderm; ac, amniotic cavity; ms, mesoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm; ecc, exocoelomic cavity; ic, intraembryonic coelomic cavity; ng, neural groove; and s, somite. Bars, 150 μm (Aa, Ba–Be, Ca, and Cb); 300 μm (Ab); and 350 μm (Ac).
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Figure 3: Developmental defects of afadin−/− embryos during early embryogenesis. (A) Gross appearance. (Aa) E7.5 embryos; (Ab) E8.5 embryos; and (Ac) E9.5 embryos: left, wild-type embryos; and right, afadin−/− embryos. (B) Histological analysis of E7.5 embryos. E7.5 embryos were subjected to staining with hematoxylin and eosin. (Ba) Wild-type embryos (transverse section); (Bb–Be) afadin−/− embryos; (Bb) transverse section of the proximal region; (Bc) transverse section of the middle region; (Bd) transverse section of the distal region; and (Be) sagittal section. (C) Histological analysis of E8.5 embryos. E8.5 embryos were subjected to staining with hematoxylin and eosin. (Ca) Wild-type embryos (transverse section); and (Cb) afadin−/− embryos (transverse section). hf, headfold; ps, primitive streak; ec, ectoplacental cone; ht, primitive heart; ee, embryonic ectoderm; ve, visceral endoderm; ac, amniotic cavity; ms, mesoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm; ecc, exocoelomic cavity; ic, intraembryonic coelomic cavity; ng, neural groove; and s, somite. Bars, 150 μm (Aa, Ba–Be, Ca, and Cb); 300 μm (Ab); and 350 μm (Ac).
Mentions: Embryos were isolated at various stages of gestation and their genotypes were determined (Table ). Distribution of each genotype examined at E7.5–E9.5 followed the Mendelian law, whereas no homozygous embryos were detected from E10.5. At E6.5, gross morphological analysis did not distinguish the homozygous embryos from the wild-type and heterozygous littermates (data not shown), indicating that implantation and egg cylinder formation occur normally in the absence of afadin. To examine the presence of residual maternal afadin, which may affect implantation and egg cylinder formation in the homozygous embryos, preimplantation embryos at E3.5 (early blastocysts) were cultured and their levels of l-afadin were determined by immunofluorescence microscopy. Of 20 embryos examined, 16 showed weak but significant staining, whereas the remaining did not show any signal (data not shown). It is most likely that the latter embryos are homozygous and that there is no residual maternal afadin in the homozygous embryos. In contrast to embryos at E6.5, it was easy to distinguish the homozygous embryos from the wild-type and heterozygous littermates in embryos at E7.5–E9.5. Compared with wild-type embryos, the architecture of the homozygous embryos was apparently distorted and reduced in size (Fig. 3, Aa–Ac). Of note, however, is that their extraembryonic regions, including ectoplacental cone and yolk sac, developed normally, indicating that the anomalies are basically restricted to embryos proper. Whereas the anterior–posterior distinction could easily be made by gross appearance, and some vascular system and blood were detectable, the homozygous embryos were always flat and short, and no landmark tissues such as heart developed at this stage. The anomalies specific for these embryos proper in afadin−/− embryos are consistent with the expression patterns of l-afadin.

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