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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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Schematic illustration of the experimental design and basic information. (A) Adenoviral expression vectors were injected into the segmental plate of stage 12 embryos. The earliest protein expression begins at 6–8 h after injection and persists at least for 3 d as depicted by the shadowing. (B) Whole-mount X-gal staining of embryos injected with adenoviral vectors encoding lacZ (AdV-lacZ) at 12, 24, and 72 h after the injection. Although the transgene is expressed in the entire segmenting mesoderm at early stages, a high level of gene expression is maintained only in myofibers at 72 h. (C and D) Wild-type and mutated N-cadherins (C) or p120ctn (D) for constructing recombinant adenoviruses. Each construct is tagged by EGFP or FLAG epitope at their COOH terminus. Amino acid sequences at the mutated portion in cN/JMΔ and cN/784AAA are shown. The underlined region was suggested to be crucial for binding to p120ctn (Thoreson et al., 2000). EC, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; CH, carboxy half domain; ARM, armadillo repeat. (E) Western blots of the recombinant N-cadherins and p120ctn were studied. Cadherin-deficient A431D cells were infected with respective viral vectors, and expressed proteins were detected with the monoclonal antibody NCD-2, which recognized the NH2-terminal region of N-cadherin. For examination of cN390Δ, the original A431 line was used for infection because this construct was unstable in cadherin-deficient cells. The immunoreactivity of cN390Δ to NCD-2 was reduced compared with that of the full-length N-cadherin (Fujimori and Takeichi, 1993), resulting in an apparently faint band. The higher MW band in the cN390Δ lane is presumably a precursor protein. p120ctn was detected with anti-Flag antibodies. Viral vectors of the same titer as used for these infections were used for in ovo infection. (F) Western blot detection of p120ctn from the immunoprecipitants of N-cad, cN/JMΔ, and cN/784AAA. These proteins were expressed in A431D cells as above, immunoprecipitated with rabbit anti–N-cadherin antibodies, and subjected to detection of p120ctn. This protein was not detected in the cN/JMΔ and cN/784AAA immunoprecipitates. Multiple p120ctn bands coprecipitated with the control N-cadherin represent isoforms of this protein (Anastasiadis and Reynolds, 2000). As a control, β-catenin was detected from all the samples.
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fig1: Schematic illustration of the experimental design and basic information. (A) Adenoviral expression vectors were injected into the segmental plate of stage 12 embryos. The earliest protein expression begins at 6–8 h after injection and persists at least for 3 d as depicted by the shadowing. (B) Whole-mount X-gal staining of embryos injected with adenoviral vectors encoding lacZ (AdV-lacZ) at 12, 24, and 72 h after the injection. Although the transgene is expressed in the entire segmenting mesoderm at early stages, a high level of gene expression is maintained only in myofibers at 72 h. (C and D) Wild-type and mutated N-cadherins (C) or p120ctn (D) for constructing recombinant adenoviruses. Each construct is tagged by EGFP or FLAG epitope at their COOH terminus. Amino acid sequences at the mutated portion in cN/JMΔ and cN/784AAA are shown. The underlined region was suggested to be crucial for binding to p120ctn (Thoreson et al., 2000). EC, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; CH, carboxy half domain; ARM, armadillo repeat. (E) Western blots of the recombinant N-cadherins and p120ctn were studied. Cadherin-deficient A431D cells were infected with respective viral vectors, and expressed proteins were detected with the monoclonal antibody NCD-2, which recognized the NH2-terminal region of N-cadherin. For examination of cN390Δ, the original A431 line was used for infection because this construct was unstable in cadherin-deficient cells. The immunoreactivity of cN390Δ to NCD-2 was reduced compared with that of the full-length N-cadherin (Fujimori and Takeichi, 1993), resulting in an apparently faint band. The higher MW band in the cN390Δ lane is presumably a precursor protein. p120ctn was detected with anti-Flag antibodies. Viral vectors of the same titer as used for these infections were used for in ovo infection. (F) Western blot detection of p120ctn from the immunoprecipitants of N-cad, cN/JMΔ, and cN/784AAA. These proteins were expressed in A431D cells as above, immunoprecipitated with rabbit anti–N-cadherin antibodies, and subjected to detection of p120ctn. This protein was not detected in the cN/JMΔ and cN/784AAA immunoprecipitates. Multiple p120ctn bands coprecipitated with the control N-cadherin represent isoforms of this protein (Anastasiadis and Reynolds, 2000). As a control, β-catenin was detected from all the samples.

Mentions: As a preliminary experiment, we injected adenoviral expression vectors encoding lacZ (AdV-lacZ) into the segmental plate of embryos at stage 12 (Fig. 1 A) and stained the enzymic products to determine the general expression profile of the cDNAs injected into this site. X-gal staining showed that the injected cDNA began to be expressed at ∼6–8 h after the injection (unpublished data). Initially, the entire population of somitic cells were stained, and then gradually the intense staining became restricted to the myotome, resulting in visualization of only an array of myofibers in each somite (Fig. 1 B). This result can be explained based on the following two facts: (a) adenoviral vectors do not replicate, and thus they become diluted in rapidly proliferating cells and (b) myotome cells enter into the postmitotic state at early developmental stages (Kahane et al., 1998). Therefore, myotome cells would be expected to maintain a high level expression of the injected cDNAs. Because of these observations, the following experiments were focused on the myotome, which likely responds most effectively to the ectopically expressed molecules.


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)

Schematic illustration of the experimental design and basic information. (A) Adenoviral expression vectors were injected into the segmental plate of stage 12 embryos. The earliest protein expression begins at 6–8 h after injection and persists at least for 3 d as depicted by the shadowing. (B) Whole-mount X-gal staining of embryos injected with adenoviral vectors encoding lacZ (AdV-lacZ) at 12, 24, and 72 h after the injection. Although the transgene is expressed in the entire segmenting mesoderm at early stages, a high level of gene expression is maintained only in myofibers at 72 h. (C and D) Wild-type and mutated N-cadherins (C) or p120ctn (D) for constructing recombinant adenoviruses. Each construct is tagged by EGFP or FLAG epitope at their COOH terminus. Amino acid sequences at the mutated portion in cN/JMΔ and cN/784AAA are shown. The underlined region was suggested to be crucial for binding to p120ctn (Thoreson et al., 2000). EC, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; CH, carboxy half domain; ARM, armadillo repeat. (E) Western blots of the recombinant N-cadherins and p120ctn were studied. Cadherin-deficient A431D cells were infected with respective viral vectors, and expressed proteins were detected with the monoclonal antibody NCD-2, which recognized the NH2-terminal region of N-cadherin. For examination of cN390Δ, the original A431 line was used for infection because this construct was unstable in cadherin-deficient cells. The immunoreactivity of cN390Δ to NCD-2 was reduced compared with that of the full-length N-cadherin (Fujimori and Takeichi, 1993), resulting in an apparently faint band. The higher MW band in the cN390Δ lane is presumably a precursor protein. p120ctn was detected with anti-Flag antibodies. Viral vectors of the same titer as used for these infections were used for in ovo infection. (F) Western blot detection of p120ctn from the immunoprecipitants of N-cad, cN/JMΔ, and cN/784AAA. These proteins were expressed in A431D cells as above, immunoprecipitated with rabbit anti–N-cadherin antibodies, and subjected to detection of p120ctn. This protein was not detected in the cN/JMΔ and cN/784AAA immunoprecipitates. Multiple p120ctn bands coprecipitated with the control N-cadherin represent isoforms of this protein (Anastasiadis and Reynolds, 2000). As a control, β-catenin was detected from all the samples.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199319&req=5

fig1: Schematic illustration of the experimental design and basic information. (A) Adenoviral expression vectors were injected into the segmental plate of stage 12 embryos. The earliest protein expression begins at 6–8 h after injection and persists at least for 3 d as depicted by the shadowing. (B) Whole-mount X-gal staining of embryos injected with adenoviral vectors encoding lacZ (AdV-lacZ) at 12, 24, and 72 h after the injection. Although the transgene is expressed in the entire segmenting mesoderm at early stages, a high level of gene expression is maintained only in myofibers at 72 h. (C and D) Wild-type and mutated N-cadherins (C) or p120ctn (D) for constructing recombinant adenoviruses. Each construct is tagged by EGFP or FLAG epitope at their COOH terminus. Amino acid sequences at the mutated portion in cN/JMΔ and cN/784AAA are shown. The underlined region was suggested to be crucial for binding to p120ctn (Thoreson et al., 2000). EC, extracellular domain; TM, transmembrane domain; JM, juxtamembrane domain; CH, carboxy half domain; ARM, armadillo repeat. (E) Western blots of the recombinant N-cadherins and p120ctn were studied. Cadherin-deficient A431D cells were infected with respective viral vectors, and expressed proteins were detected with the monoclonal antibody NCD-2, which recognized the NH2-terminal region of N-cadherin. For examination of cN390Δ, the original A431 line was used for infection because this construct was unstable in cadherin-deficient cells. The immunoreactivity of cN390Δ to NCD-2 was reduced compared with that of the full-length N-cadherin (Fujimori and Takeichi, 1993), resulting in an apparently faint band. The higher MW band in the cN390Δ lane is presumably a precursor protein. p120ctn was detected with anti-Flag antibodies. Viral vectors of the same titer as used for these infections were used for in ovo infection. (F) Western blot detection of p120ctn from the immunoprecipitants of N-cad, cN/JMΔ, and cN/784AAA. These proteins were expressed in A431D cells as above, immunoprecipitated with rabbit anti–N-cadherin antibodies, and subjected to detection of p120ctn. This protein was not detected in the cN/JMΔ and cN/784AAA immunoprecipitates. Multiple p120ctn bands coprecipitated with the control N-cadherin represent isoforms of this protein (Anastasiadis and Reynolds, 2000). As a control, β-catenin was detected from all the samples.
Mentions: As a preliminary experiment, we injected adenoviral expression vectors encoding lacZ (AdV-lacZ) into the segmental plate of embryos at stage 12 (Fig. 1 A) and stained the enzymic products to determine the general expression profile of the cDNAs injected into this site. X-gal staining showed that the injected cDNA began to be expressed at ∼6–8 h after the injection (unpublished data). Initially, the entire population of somitic cells were stained, and then gradually the intense staining became restricted to the myotome, resulting in visualization of only an array of myofibers in each somite (Fig. 1 B). This result can be explained based on the following two facts: (a) adenoviral vectors do not replicate, and thus they become diluted in rapidly proliferating cells and (b) myotome cells enter into the postmitotic state at early developmental stages (Kahane et al., 1998). Therefore, myotome cells would be expected to maintain a high level expression of the injected cDNAs. Because of these observations, the following experiments were focused on the myotome, which likely responds most effectively to the ectopically expressed molecules.

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