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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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Disorganized cell–cell junctions of the embryonic ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin and anti–ZO-1 antibodies. (A and B) Wild-type embryos; (C–F) afadin−/− embryos; (C) proximal region; (D) high magnification of the indicated box D in Cc; (E) high magnification of the indicated box E in Cc; and (F) distal region. (Aa, Ba, Ca, Da, Ea, and Fa) E-cadherin; (Ab, Bb, Cb, Db, Eb, and Fb) ZO-1; and (Ac, Bc, Cc, Dc, Ec, and Fc) merge. Asterisks indicate a typical region where E-cadherin is concentrated at the junctional complex regions. ps, primitive streak; ac, amniotic cavity; ne, neuroepithelium; ms, mesoderm; ve, visceral endoderm; ng, neural groove; nf, neural fold; ee, embryonic ectoderm; and cm, cell mass. Bars, 30 μm.
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Figure 6: Disorganized cell–cell junctions of the embryonic ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin and anti–ZO-1 antibodies. (A and B) Wild-type embryos; (C–F) afadin−/− embryos; (C) proximal region; (D) high magnification of the indicated box D in Cc; (E) high magnification of the indicated box E in Cc; and (F) distal region. (Aa, Ba, Ca, Da, Ea, and Fa) E-cadherin; (Ab, Bb, Cb, Db, Eb, and Fb) ZO-1; and (Ac, Bc, Cc, Dc, Ec, and Fc) merge. Asterisks indicate a typical region where E-cadherin is concentrated at the junctional complex regions. ps, primitive streak; ac, amniotic cavity; ne, neuroepithelium; ms, mesoderm; ve, visceral endoderm; ng, neural groove; nf, neural fold; ee, embryonic ectoderm; and cm, cell mass. Bars, 30 μm.

Mentions: To further dissect the developmental defects of afadin−/− embryos, we next investigated expression of E-cadherin and mesoderm markers, including T, PDGFRα, and Flk1 at E7.5. Consistent with an earlier observation (Wilkinson et al. 1990), T was highly expressed in the primitive streak and its nascent mesoderm in wild-type embryos (Fig. 4, Aa and Ab). In afadin−/− embryos, the T-positive area appeared to be divided into two portions that corresponded to the most posterior regions of the two-layered ectoderm (Fig. 4, Ba–Bd). At the primitive streak stage, E-cadherin is expressed in the entire embryonic ectoderm (Takeichi 1988). PDGFRα is expressed in the paraxial mesoderm (Takakura et al. 1997), and Flk1 is expressed in the proximal lateral mesoderm and the extraembryonic mesoderm (Kataoka et al. 1997). After completion of exfoliation from the primitive streak, E-cadherin was downregulated, and PDGFRα and Flk1 were expressed in the mesodermal cells (Fig. 5, Aa–Ac and Ea–Ec). In afadin−/− embryos, E-cadherin–negative and PDGFRα-positive cells were detected at the space between the ectoderm and the endoderm (Fig. 5, Ba–Bc, Ca–Cc, and Da–Dc). These cells corresponded to the mesodermal cells of the paraxial region. At the proximal region of the primitive streak, E-cadherin–positive cells were jammed at the space between the ectoderm and the endoderm (Fig. 5, Ba, Bc, Fa, and Fc; see also Fig. 6, Ca, Cc, Da, and Dc). The staining for E-cadherin clearly demonstrated that the E-cadherin–positive ectoderm was invaginated from the posterior region toward the amniotic side (Fig. 5, Ca and Cc). The most posterior region of the two-layered ectoderm corresponded to the area positive for T (Fig. 5, Ca and Cc; see also Fig. 4 Bc). E-cadherin–negative and PDGFRα-positive cells were detected at the space surrounded by the invaginated ectoderm. These cells appeared to migrate from the primitive streak. At the regions corresponding to neural fold/groove (from the anterior region to the distal region), the multilayered cells were E-cadherin–positive (Fig. 5, Ba, Bc, Ca, Cc, Da, and Dc). At the distal region, some cells in the cell mass expressed not only E-cadherin but also PDGFRα (Fig. 5, Da–Dc). This cell mass was surrounded by a layer of the ectodermal cells that were E-cadherin–positive and PDGFRα-negative. Similar observations were obtained with the double staining for E-cadherin and Flk1, except that Flk1 was not expressed in the cell mass (Fig. 5, E–H). These observations strongly suggest that the major histological basis of the developmental defects of afadin−/− mice is disorganization of the embryonic ectoderm, and that distorted placement of various cell lineages is the secondary outcome of this disorganization.


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 cell–cell junctions of the embryonic ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin and anti–ZO-1 antibodies. (A and B) Wild-type embryos; (C–F) afadin−/− embryos; (C) proximal region; (D) high magnification of the indicated box D in Cc; (E) high magnification of the indicated box E in Cc; and (F) distal region. (Aa, Ba, Ca, Da, Ea, and Fa) E-cadherin; (Ab, Bb, Cb, Db, Eb, and Fb) ZO-1; and (Ac, Bc, Cc, Dc, Ec, and Fc) merge. Asterisks indicate a typical region where E-cadherin is concentrated at the junctional complex regions. ps, primitive streak; ac, amniotic cavity; ne, neuroepithelium; ms, mesoderm; ve, visceral endoderm; ng, neural groove; nf, neural fold; ee, embryonic ectoderm; and cm, cell mass. Bars, 30 μm.
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Figure 6: Disorganized cell–cell junctions of the embryonic ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin and anti–ZO-1 antibodies. (A and B) Wild-type embryos; (C–F) afadin−/− embryos; (C) proximal region; (D) high magnification of the indicated box D in Cc; (E) high magnification of the indicated box E in Cc; and (F) distal region. (Aa, Ba, Ca, Da, Ea, and Fa) E-cadherin; (Ab, Bb, Cb, Db, Eb, and Fb) ZO-1; and (Ac, Bc, Cc, Dc, Ec, and Fc) merge. Asterisks indicate a typical region where E-cadherin is concentrated at the junctional complex regions. ps, primitive streak; ac, amniotic cavity; ne, neuroepithelium; ms, mesoderm; ve, visceral endoderm; ng, neural groove; nf, neural fold; ee, embryonic ectoderm; and cm, cell mass. Bars, 30 μm.
Mentions: To further dissect the developmental defects of afadin−/− embryos, we next investigated expression of E-cadherin and mesoderm markers, including T, PDGFRα, and Flk1 at E7.5. Consistent with an earlier observation (Wilkinson et al. 1990), T was highly expressed in the primitive streak and its nascent mesoderm in wild-type embryos (Fig. 4, Aa and Ab). In afadin−/− embryos, the T-positive area appeared to be divided into two portions that corresponded to the most posterior regions of the two-layered ectoderm (Fig. 4, Ba–Bd). At the primitive streak stage, E-cadherin is expressed in the entire embryonic ectoderm (Takeichi 1988). PDGFRα is expressed in the paraxial mesoderm (Takakura et al. 1997), and Flk1 is expressed in the proximal lateral mesoderm and the extraembryonic mesoderm (Kataoka et al. 1997). After completion of exfoliation from the primitive streak, E-cadherin was downregulated, and PDGFRα and Flk1 were expressed in the mesodermal cells (Fig. 5, Aa–Ac and Ea–Ec). In afadin−/− embryos, E-cadherin–negative and PDGFRα-positive cells were detected at the space between the ectoderm and the endoderm (Fig. 5, Ba–Bc, Ca–Cc, and Da–Dc). These cells corresponded to the mesodermal cells of the paraxial region. At the proximal region of the primitive streak, E-cadherin–positive cells were jammed at the space between the ectoderm and the endoderm (Fig. 5, Ba, Bc, Fa, and Fc; see also Fig. 6, Ca, Cc, Da, and Dc). The staining for E-cadherin clearly demonstrated that the E-cadherin–positive ectoderm was invaginated from the posterior region toward the amniotic side (Fig. 5, Ca and Cc). The most posterior region of the two-layered ectoderm corresponded to the area positive for T (Fig. 5, Ca and Cc; see also Fig. 4 Bc). E-cadherin–negative and PDGFRα-positive cells were detected at the space surrounded by the invaginated ectoderm. These cells appeared to migrate from the primitive streak. At the regions corresponding to neural fold/groove (from the anterior region to the distal region), the multilayered cells were E-cadherin–positive (Fig. 5, Ba, Bc, Ca, Cc, Da, and Dc). At the distal region, some cells in the cell mass expressed not only E-cadherin but also PDGFRα (Fig. 5, Da–Dc). This cell mass was surrounded by a layer of the ectodermal cells that were E-cadherin–positive and PDGFRα-negative. Similar observations were obtained with the double staining for E-cadherin and Flk1, except that Flk1 was not expressed in the cell mass (Fig. 5, E–H). These observations strongly suggest that the major histological basis of the developmental defects of afadin−/− mice is disorganization of the embryonic ectoderm, and that distorted placement of various cell lineages is the secondary outcome of this disorganization.

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