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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 ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin antibody and the anti-PDGFRα or anti-Flk1 antibody for immunofluorescence microscopy. (A and E) Wild-type embryos; (B–D and F–H) afadin−/− embryos; (B and F) proximal region; (C and G) middle region; (D and H) distal region; (A–D) double staining with the anti–E-cadherin and anti-PDGFRα antibodies; (E–H) double staining with the anti–E-cadherin and anti-Flk1 antibodies. (Aa, Ba, Ca, Da, Ea, Fa, Ga, and Ha) E-cadherin; (Ab, Bb, Cb, and Db) PDGFRα; (Eb, Fb, Gb, and Hb) Flk1; and (Ac, Bc, Cc, Dc, Ec, Fc, Gc, and Hc) merge. ac, amniotic cavity; ms, mesoderm; ne, neuroepithelium; ve, visceral endoderm; ps, primitive streak; ee, embryonic ectoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm. Bars, 60 μm.
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Figure 5: Disorganized ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin antibody and the anti-PDGFRα or anti-Flk1 antibody for immunofluorescence microscopy. (A and E) Wild-type embryos; (B–D and F–H) afadin−/− embryos; (B and F) proximal region; (C and G) middle region; (D and H) distal region; (A–D) double staining with the anti–E-cadherin and anti-PDGFRα antibodies; (E–H) double staining with the anti–E-cadherin and anti-Flk1 antibodies. (Aa, Ba, Ca, Da, Ea, Fa, Ga, and Ha) E-cadherin; (Ab, Bb, Cb, and Db) PDGFRα; (Eb, Fb, Gb, and Hb) Flk1; and (Ac, Bc, Cc, Dc, Ec, Fc, Gc, and Hc) merge. ac, amniotic cavity; ms, mesoderm; ne, neuroepithelium; ve, visceral endoderm; ps, primitive streak; ee, embryonic ectoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm. Bars, 60 μm.

Mentions: In wild-type embryos at E7.5, delamination of mesodermal cells from the primitive streak was undertaken in a strictly polarized manner, thereby recruiting mesodermal cells into the space between the embryonic ectoderm and the visceral endoderm (Fig. 3 Ba). Of importance is that the integrity of epithelial structures of the ectoderm, including the primitive streak and neural groove, is maintained even under the stimulation to induce delamination. In afadin−/− embryos at E7.5, generation of mesodermal cells at this space did occur, indicating that mesoderm induction itself occurs normally (Fig. 3, Bb–Be). However, these mutant embryos had the wider space between the ectoderm and the endoderm, where more mesodermal cells were observed compared with wild-type embryos. Ectodermal cells of afadin−/− embryos appeared flat or cuboid, and their polarized epithelial structures appeared to be entirely impaired (Fig. 3, Bb–Be). At the region corresponding to neural fold/groove (from the anterior region to the distal region), the ectoderm became multilayered and appeared as a cell mass (Fig. 3, Bb–Be). At the posterior region corresponding to the primitive streak, the ectoderm was invaginated toward the amniotic side (Fig. 3, Bc and Be; see also Fig. 4 Bc, and Fig. 5, Ca, Cc, Ga, and Gc). The invaginated ectoderm often ran along with the ectoderm of the lateral region, resulting in the appearance of two layers. The space between the two layers corresponded to the amniotic cavity, which was compressed to an inverted U-shape. Cells were detected at the space surrounded by the invaginated ectoderm. Furthermore, in afadin−/− embryos, amniotic and exocoelomic cavities did not develop normally and formation of allantois was not observed (Fig. 3 Be). In contrast to the severe defects of the embryonic ectoderm, a single-layered epithelial structure in the endoderm remained intact (Fig. 3, Bb–Be). Hence, all of these phenotypes may be an outcome of abnormal progression of amniotic and chorionic membrane formation from the most proximal region of the primitive streak. In wild-type embryos at E8.5, tissues with epithelial structures, such as neural groove, intraembryonic coelomic cavity, and somites, were clearly evident (Fig. 3 Ca). In afadin−/− embryos, consistent with histological findings showing generation of mesodermal cells, gross appearance showed that somite-like blocks and some vascular structures were detected (data not shown), although no epithelial structure of somites was established (Fig. 3 Cb). Formation of neither neural tube nor heart was observed.


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 afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin antibody and the anti-PDGFRα or anti-Flk1 antibody for immunofluorescence microscopy. (A and E) Wild-type embryos; (B–D and F–H) afadin−/− embryos; (B and F) proximal region; (C and G) middle region; (D and H) distal region; (A–D) double staining with the anti–E-cadherin and anti-PDGFRα antibodies; (E–H) double staining with the anti–E-cadherin and anti-Flk1 antibodies. (Aa, Ba, Ca, Da, Ea, Fa, Ga, and Ha) E-cadherin; (Ab, Bb, Cb, and Db) PDGFRα; (Eb, Fb, Gb, and Hb) Flk1; and (Ac, Bc, Cc, Dc, Ec, Fc, Gc, and Hc) merge. ac, amniotic cavity; ms, mesoderm; ne, neuroepithelium; ve, visceral endoderm; ps, primitive streak; ee, embryonic ectoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm. Bars, 60 μm.
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Related In: Results  -  Collection

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Figure 5: Disorganized ectoderm in afadin−/− embryos. Transverse sections of E7.5 embryos were doubly stained with the anti–E-cadherin antibody and the anti-PDGFRα or anti-Flk1 antibody for immunofluorescence microscopy. (A and E) Wild-type embryos; (B–D and F–H) afadin−/− embryos; (B and F) proximal region; (C and G) middle region; (D and H) distal region; (A–D) double staining with the anti–E-cadherin and anti-PDGFRα antibodies; (E–H) double staining with the anti–E-cadherin and anti-Flk1 antibodies. (Aa, Ba, Ca, Da, Ea, Fa, Ga, and Ha) E-cadherin; (Ab, Bb, Cb, and Db) PDGFRα; (Eb, Fb, Gb, and Hb) Flk1; and (Ac, Bc, Cc, Dc, Ec, Fc, Gc, and Hc) merge. ac, amniotic cavity; ms, mesoderm; ne, neuroepithelium; ve, visceral endoderm; ps, primitive streak; ee, embryonic ectoderm; cm, cell mass; lee, lateral region of the embryonic ectoderm; iee, invaginated embryonic ectoderm. Bars, 60 μm.
Mentions: In wild-type embryos at E7.5, delamination of mesodermal cells from the primitive streak was undertaken in a strictly polarized manner, thereby recruiting mesodermal cells into the space between the embryonic ectoderm and the visceral endoderm (Fig. 3 Ba). Of importance is that the integrity of epithelial structures of the ectoderm, including the primitive streak and neural groove, is maintained even under the stimulation to induce delamination. In afadin−/− embryos at E7.5, generation of mesodermal cells at this space did occur, indicating that mesoderm induction itself occurs normally (Fig. 3, Bb–Be). However, these mutant embryos had the wider space between the ectoderm and the endoderm, where more mesodermal cells were observed compared with wild-type embryos. Ectodermal cells of afadin−/− embryos appeared flat or cuboid, and their polarized epithelial structures appeared to be entirely impaired (Fig. 3, Bb–Be). At the region corresponding to neural fold/groove (from the anterior region to the distal region), the ectoderm became multilayered and appeared as a cell mass (Fig. 3, Bb–Be). At the posterior region corresponding to the primitive streak, the ectoderm was invaginated toward the amniotic side (Fig. 3, Bc and Be; see also Fig. 4 Bc, and Fig. 5, Ca, Cc, Ga, and Gc). The invaginated ectoderm often ran along with the ectoderm of the lateral region, resulting in the appearance of two layers. The space between the two layers corresponded to the amniotic cavity, which was compressed to an inverted U-shape. Cells were detected at the space surrounded by the invaginated ectoderm. Furthermore, in afadin−/− embryos, amniotic and exocoelomic cavities did not develop normally and formation of allantois was not observed (Fig. 3 Be). In contrast to the severe defects of the embryonic ectoderm, a single-layered epithelial structure in the endoderm remained intact (Fig. 3, Bb–Be). Hence, all of these phenotypes may be an outcome of abnormal progression of amniotic and chorionic membrane formation from the most proximal region of the primitive streak. In wild-type embryos at E8.5, tissues with epithelial structures, such as neural groove, intraembryonic coelomic cavity, and somites, were clearly evident (Fig. 3 Ca). In afadin−/− embryos, consistent with histological findings showing generation of mesodermal cells, gross appearance showed that somite-like blocks and some vascular structures were detected (data not shown), although no epithelial structure of somites was established (Fig. 3 Cb). Formation of neither neural tube nor heart was observed.

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