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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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Generation of afadin−/− ES cells. (A) Restriction maps of the afadin+/− allele, the second targeting vector, and the afadin−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; and X, XbaI. (B) Southern blot analysis. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Western blot analysis. A cell lysate of ES cells, simple EBs, or cystic EBs (20 μg of protein each) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin mAb or the mAb recognizing both l- and s-afadins. sEBs, simple EBs. cEBs, cystic EBs. Black arrowheads indicate l-afadin and white arrowhead indicates s-afadin.
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Figure 7: Generation of afadin−/− ES cells. (A) Restriction maps of the afadin+/− allele, the second targeting vector, and the afadin−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; and X, XbaI. (B) Southern blot analysis. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Western blot analysis. A cell lysate of ES cells, simple EBs, or cystic EBs (20 μg of protein each) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin mAb or the mAb recognizing both l- and s-afadins. sEBs, simple EBs. cEBs, cystic EBs. Black arrowheads indicate l-afadin and white arrowhead indicates s-afadin.

Mentions: To determine whether or not the defects in afadin−/− embryos can be reproduced in a simpler model system, we took advantage of EB formation of ES cells where development of two-layered epithelial structures and subsequent mesoderm induction from the inner layer are shown to be reproduced in vitro (Doetschman et al. 1985; Robertson 1987; Rudnicki and McBurney 1987). For this purpose, we first established an afadin−/− ES cell line by introducing another targeting vector harboring puromycin-resistant gene (Fig. 7 A). This targeting vector was introduced into afadin+/− ES cells (clone A46). ES cells were subjected to selection using puromycin. Southern blot analysis showed that three clones (B3, B8, and B103) resistant to puromycin underwent gene conversion, resulting in disruption of both alleles (Fig. 7 B). Western blot analysis using the anti–l-afadin (rat, aa 1814–1829) mAb and the mAb recognizing both l- and s-afadins (human, aa 1091–1233) revealed the loss of afadin in afadin−/− ES cells and EBs (Fig. 7 C). Similar results were obtained with the polyclonal antibody recognizing both l- and s-afadins (rat, aa 577–592) (data not shown). Western blot analysis also revealed that l-afadin was a major expressed variant in wild-type ES cells and EBs, and that s-afadin was hardly detected (Fig. 7 C). Reverse transcription PCR analysis using the primer set corresponding to aa 188–316 of mouse afadin showed the loss of the afadin mRNA in afadin−/− ES cells (data not shown). The three independent clones of afadin−/− ES cells showed the same growth rate with undifferentiated morphology as wild-type ES cells did (data not shown). Since these clones showed the same phenotypes as far as examined, the data obtained from clone B3 were represented below.


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)

Generation of afadin−/− ES cells. (A) Restriction maps of the afadin+/− allele, the second targeting vector, and the afadin−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; and X, XbaI. (B) Southern blot analysis. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Western blot analysis. A cell lysate of ES cells, simple EBs, or cystic EBs (20 μg of protein each) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin mAb or the mAb recognizing both l- and s-afadins. sEBs, simple EBs. cEBs, cystic EBs. Black arrowheads indicate l-afadin and white arrowhead indicates s-afadin.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Generation of afadin−/− ES cells. (A) Restriction maps of the afadin+/− allele, the second targeting vector, and the afadin−/− allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; and X, XbaI. (B) Southern blot analysis. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Western blot analysis. A cell lysate of ES cells, simple EBs, or cystic EBs (20 μg of protein each) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin mAb or the mAb recognizing both l- and s-afadins. sEBs, simple EBs. cEBs, cystic EBs. Black arrowheads indicate l-afadin and white arrowhead indicates s-afadin.
Mentions: To determine whether or not the defects in afadin−/− embryos can be reproduced in a simpler model system, we took advantage of EB formation of ES cells where development of two-layered epithelial structures and subsequent mesoderm induction from the inner layer are shown to be reproduced in vitro (Doetschman et al. 1985; Robertson 1987; Rudnicki and McBurney 1987). For this purpose, we first established an afadin−/− ES cell line by introducing another targeting vector harboring puromycin-resistant gene (Fig. 7 A). This targeting vector was introduced into afadin+/− ES cells (clone A46). ES cells were subjected to selection using puromycin. Southern blot analysis showed that three clones (B3, B8, and B103) resistant to puromycin underwent gene conversion, resulting in disruption of both alleles (Fig. 7 B). Western blot analysis using the anti–l-afadin (rat, aa 1814–1829) mAb and the mAb recognizing both l- and s-afadins (human, aa 1091–1233) revealed the loss of afadin in afadin−/− ES cells and EBs (Fig. 7 C). Similar results were obtained with the polyclonal antibody recognizing both l- and s-afadins (rat, aa 577–592) (data not shown). Western blot analysis also revealed that l-afadin was a major expressed variant in wild-type ES cells and EBs, and that s-afadin was hardly detected (Fig. 7 C). Reverse transcription PCR analysis using the primer set corresponding to aa 188–316 of mouse afadin showed the loss of the afadin mRNA in afadin−/− ES cells (data not shown). The three independent clones of afadin−/− ES cells showed the same growth rate with undifferentiated morphology as wild-type ES cells did (data not shown). Since these clones showed the same phenotypes as far as examined, the data obtained from clone B3 were represented below.

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