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Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development.

Horikawa K, Takeichi M - J. Cell Biol. (2001)

Bottom Line: However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development.Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries.Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain-dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain-deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.

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Expression of the JM domain–deficient cadherin affects the morphology of myotomes. Embryos were coinjected with AdV-lacZ and AdV-Ncad (A and C) or AdV-cN/JM(−) (B and D) and subjected to whole-mount X-gal staining at 72 h after the injection. Boxed regions in A and B are enlarged in C and D, respectively. Embryos were oriented with their rostral aspect to the right. (E and F) Transverse sections of embryos injected with AdV-Ncad (E) or AdV-cN/JM(−) (F) were double immunostained for the tag-bearing cadherin (red) and β-catenin (green) at 72 h after the injection. Myotome cells expressing the JM(−) cadherin are clumped in a region at the level of motor axon exit. (G and H) Embryos injected with AdV-Ncad (G) or AdV-cN/JM(−) (H) were immunostained as whole-mount samples for a muscle differentiation marker, MHC, at 36 h after the injection. Expression of neither control nor JM(−) cadherin did not affect muscle differentiation. Bars, 500 μm.
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fig2: Expression of the JM domain–deficient cadherin affects the morphology of myotomes. Embryos were coinjected with AdV-lacZ and AdV-Ncad (A and C) or AdV-cN/JM(−) (B and D) and subjected to whole-mount X-gal staining at 72 h after the injection. Boxed regions in A and B are enlarged in C and D, respectively. Embryos were oriented with their rostral aspect to the right. (E and F) Transverse sections of embryos injected with AdV-Ncad (E) or AdV-cN/JM(−) (F) were double immunostained for the tag-bearing cadherin (red) and β-catenin (green) at 72 h after the injection. Myotome cells expressing the JM(−) cadherin are clumped in a region at the level of motor axon exit. (G and H) Embryos injected with AdV-Ncad (G) or AdV-cN/JM(−) (H) were immunostained as whole-mount samples for a muscle differentiation marker, MHC, at 36 h after the injection. Expression of neither control nor JM(−) cadherin did not affect muscle differentiation. Bars, 500 μm.

Mentions: We constructed adenoviral expression vectors for a series of N-cadherin mutants (Fig. 1 C) whose protein products, expressed in cell lines, were shown in Fig. 1 E. We first injected the one encoding the full-length N-cadherin (AdV-Ncad) or a mutant N-cadherin without the JM domain (AdV-cN/JM[−]) into the segmental plate together with AdV-lacZ. Whole-mount X-gal staining of these embryos, incubated for 3 d after injection, showed that in AdV-Ncad–injected embryos LacZ-positive myofibers became distributed normally, whereas in AdV-cN/JM(−)–injected embryos the stain signals clustered abnormally at a mid-lateral position of the trunk (Fig. 2, A–D), indicating that the DV myotome expansion was prohibited. Then, we examined sections of such treated embryos that had been doubly stained for the tag attached to the cadherin constructs and for β-catenin (Fig. 2, E and F). Tag-positive cells in AdV-Ncad–injected embryos were distributed in a pattern characteristic of the normal myotome. In contrast, in AdV-cN/JM(−)–injected embryos tag-labeled cells were clumped in a region at the level of motor axon exit, consistent with the above whole-mount observations. The staining for β-catenin was used to visualize the overall tissue architecture, and by it most tissues appeared normal with the exception of the myotome in the AdV-cN/JM(−)–injected embryos.


Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development.

Horikawa K, Takeichi M - J. Cell Biol. (2001)

Expression of the JM domain–deficient cadherin affects the morphology of myotomes. Embryos were coinjected with AdV-lacZ and AdV-Ncad (A and C) or AdV-cN/JM(−) (B and D) and subjected to whole-mount X-gal staining at 72 h after the injection. Boxed regions in A and B are enlarged in C and D, respectively. Embryos were oriented with their rostral aspect to the right. (E and F) Transverse sections of embryos injected with AdV-Ncad (E) or AdV-cN/JM(−) (F) were double immunostained for the tag-bearing cadherin (red) and β-catenin (green) at 72 h after the injection. Myotome cells expressing the JM(−) cadherin are clumped in a region at the level of motor axon exit. (G and H) Embryos injected with AdV-Ncad (G) or AdV-cN/JM(−) (H) were immunostained as whole-mount samples for a muscle differentiation marker, MHC, at 36 h after the injection. Expression of neither control nor JM(−) cadherin did not affect muscle differentiation. Bars, 500 μm.
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Related In: Results  -  Collection

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fig2: Expression of the JM domain–deficient cadherin affects the morphology of myotomes. Embryos were coinjected with AdV-lacZ and AdV-Ncad (A and C) or AdV-cN/JM(−) (B and D) and subjected to whole-mount X-gal staining at 72 h after the injection. Boxed regions in A and B are enlarged in C and D, respectively. Embryos were oriented with their rostral aspect to the right. (E and F) Transverse sections of embryos injected with AdV-Ncad (E) or AdV-cN/JM(−) (F) were double immunostained for the tag-bearing cadherin (red) and β-catenin (green) at 72 h after the injection. Myotome cells expressing the JM(−) cadherin are clumped in a region at the level of motor axon exit. (G and H) Embryos injected with AdV-Ncad (G) or AdV-cN/JM(−) (H) were immunostained as whole-mount samples for a muscle differentiation marker, MHC, at 36 h after the injection. Expression of neither control nor JM(−) cadherin did not affect muscle differentiation. Bars, 500 μm.
Mentions: We constructed adenoviral expression vectors for a series of N-cadherin mutants (Fig. 1 C) whose protein products, expressed in cell lines, were shown in Fig. 1 E. We first injected the one encoding the full-length N-cadherin (AdV-Ncad) or a mutant N-cadherin without the JM domain (AdV-cN/JM[−]) into the segmental plate together with AdV-lacZ. Whole-mount X-gal staining of these embryos, incubated for 3 d after injection, showed that in AdV-Ncad–injected embryos LacZ-positive myofibers became distributed normally, whereas in AdV-cN/JM(−)–injected embryos the stain signals clustered abnormally at a mid-lateral position of the trunk (Fig. 2, A–D), indicating that the DV myotome expansion was prohibited. Then, we examined sections of such treated embryos that had been doubly stained for the tag attached to the cadherin constructs and for β-catenin (Fig. 2, E and F). Tag-positive cells in AdV-Ncad–injected embryos were distributed in a pattern characteristic of the normal myotome. In contrast, in AdV-cN/JM(−)–injected embryos tag-labeled cells were clumped in a region at the level of motor axon exit, consistent with the above whole-mount observations. The staining for β-catenin was used to visualize the overall tissue architecture, and by it most tissues appeared normal with the exception of the myotome in the AdV-cN/JM(−)–injected embryos.

Bottom Line: However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development.Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries.Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain-dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain-deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.

Show MeSH
Related in: MedlinePlus