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Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

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Inhibition of activin-induced morphogenetic movement of animal cap explants by mAb AA5. (A) Inhibition of the  elongation of activin-induced animal caps by mAb AA5. Xenopus animal caps were incubated with or without activin and  treated with mAb AA5 Fab (1 μg/ml), mAb 6B6 Fab (1 μg/ml),  or nonimmune mouse IgG Fab (1 μg/ml). (B) Frequency of inhibition of elongation by mAb AA5. Activin-induced elongation  was plotted as a percentage of the total. n = total animal caps analyzed. Any explant exhibiting a discernible protrusion was  scored as elongated. (C) mAb AA5 did not inhibit the induction  of expression of mesodermal gene markers in response to activin.  Animal caps were incubated with or without activin and incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse  IgG Fab (1 μg/ml) until gastrula stage 10.5. Total RNA was harvested and mRNA was analyzed by RT-PCR for the presence of  the indicated transcripts. RNA from whole embryos (E) provides  a positive control. The −RT lane is identical to the embryo lane,  except reverse transcriptase was omitted and serves as a negative  control. EF-1, ubiquitously expressed, is a loading control.  Brachyury is a marker of general mesoderm. Goosecoid is a  marker of dorsal mesoderm.
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Figure 3: Inhibition of activin-induced morphogenetic movement of animal cap explants by mAb AA5. (A) Inhibition of the elongation of activin-induced animal caps by mAb AA5. Xenopus animal caps were incubated with or without activin and treated with mAb AA5 Fab (1 μg/ml), mAb 6B6 Fab (1 μg/ml), or nonimmune mouse IgG Fab (1 μg/ml). (B) Frequency of inhibition of elongation by mAb AA5. Activin-induced elongation was plotted as a percentage of the total. n = total animal caps analyzed. Any explant exhibiting a discernible protrusion was scored as elongated. (C) mAb AA5 did not inhibit the induction of expression of mesodermal gene markers in response to activin. Animal caps were incubated with or without activin and incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse IgG Fab (1 μg/ml) until gastrula stage 10.5. Total RNA was harvested and mRNA was analyzed by RT-PCR for the presence of the indicated transcripts. RNA from whole embryos (E) provides a positive control. The −RT lane is identical to the embryo lane, except reverse transcriptase was omitted and serves as a negative control. EF-1, ubiquitously expressed, is a loading control. Brachyury is a marker of general mesoderm. Goosecoid is a marker of dorsal mesoderm.

Mentions: Activin induces Xenopus animal cap explants to elongate as a result of convergent extension movements (Symes and Smith, 1987; Brieher and Gumbiner, 1994) (Fig. 3). We proposed that the activin-induced decrease in C-cadherin–mediated adhesion is necessary for tissue elongation (Brieher and Gumbiner, 1994). To test this hypothesis, we asked whether mAb AA5, which maintains high levels of C-cadherin–mediated adhesion even after activin treatment, would affect animal cap elongation (Fig. 3, A and B). As described previously, activin induced ∼95% of the animal caps to elongate significantly, forming asymmetric structures. In the absence of activin, all of the animal caps form nearly spherical structures. mAb AA5 strongly inhibited animal cap elongation in response to activin (Fig. 3 A, far right). Only ∼30% of the animal caps formed asymmetric structures that could be scored as elongated (Fig. 3 B), and even these were relatively stunted compared to the nonimmune IgG control. Although the majority of the mAb AA5-treated caps did not elongate significantly, they appeared “lumpier” than the smooth spherical structures formed in the absence of activin, suggesting that mAb AA5 did not completely block all morphogenetic movement. A different mAb that inhibits C-cadherin–mediated adhesion, 6B6, did not block elongation to the same extent as mAb AA5, even though it did affect the morphology of the elongated caps. Thus, preventing the activin-induced decrease in C-cadherin–mediated adhesion significantly inhibited the morphogenetic elongation of the tissue, indicating that the change in C-cadherin–mediated adhesion is required for the process.


Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Inhibition of activin-induced morphogenetic movement of animal cap explants by mAb AA5. (A) Inhibition of the  elongation of activin-induced animal caps by mAb AA5. Xenopus animal caps were incubated with or without activin and  treated with mAb AA5 Fab (1 μg/ml), mAb 6B6 Fab (1 μg/ml),  or nonimmune mouse IgG Fab (1 μg/ml). (B) Frequency of inhibition of elongation by mAb AA5. Activin-induced elongation  was plotted as a percentage of the total. n = total animal caps analyzed. Any explant exhibiting a discernible protrusion was  scored as elongated. (C) mAb AA5 did not inhibit the induction  of expression of mesodermal gene markers in response to activin.  Animal caps were incubated with or without activin and incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse  IgG Fab (1 μg/ml) until gastrula stage 10.5. Total RNA was harvested and mRNA was analyzed by RT-PCR for the presence of  the indicated transcripts. RNA from whole embryos (E) provides  a positive control. The −RT lane is identical to the embryo lane,  except reverse transcriptase was omitted and serves as a negative  control. EF-1, ubiquitously expressed, is a loading control.  Brachyury is a marker of general mesoderm. Goosecoid is a  marker of dorsal mesoderm.
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Related In: Results  -  Collection

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Figure 3: Inhibition of activin-induced morphogenetic movement of animal cap explants by mAb AA5. (A) Inhibition of the elongation of activin-induced animal caps by mAb AA5. Xenopus animal caps were incubated with or without activin and treated with mAb AA5 Fab (1 μg/ml), mAb 6B6 Fab (1 μg/ml), or nonimmune mouse IgG Fab (1 μg/ml). (B) Frequency of inhibition of elongation by mAb AA5. Activin-induced elongation was plotted as a percentage of the total. n = total animal caps analyzed. Any explant exhibiting a discernible protrusion was scored as elongated. (C) mAb AA5 did not inhibit the induction of expression of mesodermal gene markers in response to activin. Animal caps were incubated with or without activin and incubated either with mAb AA5 Fab (1 μg/ml) or nonimmune mouse IgG Fab (1 μg/ml) until gastrula stage 10.5. Total RNA was harvested and mRNA was analyzed by RT-PCR for the presence of the indicated transcripts. RNA from whole embryos (E) provides a positive control. The −RT lane is identical to the embryo lane, except reverse transcriptase was omitted and serves as a negative control. EF-1, ubiquitously expressed, is a loading control. Brachyury is a marker of general mesoderm. Goosecoid is a marker of dorsal mesoderm.
Mentions: Activin induces Xenopus animal cap explants to elongate as a result of convergent extension movements (Symes and Smith, 1987; Brieher and Gumbiner, 1994) (Fig. 3). We proposed that the activin-induced decrease in C-cadherin–mediated adhesion is necessary for tissue elongation (Brieher and Gumbiner, 1994). To test this hypothesis, we asked whether mAb AA5, which maintains high levels of C-cadherin–mediated adhesion even after activin treatment, would affect animal cap elongation (Fig. 3, A and B). As described previously, activin induced ∼95% of the animal caps to elongate significantly, forming asymmetric structures. In the absence of activin, all of the animal caps form nearly spherical structures. mAb AA5 strongly inhibited animal cap elongation in response to activin (Fig. 3 A, far right). Only ∼30% of the animal caps formed asymmetric structures that could be scored as elongated (Fig. 3 B), and even these were relatively stunted compared to the nonimmune IgG control. Although the majority of the mAb AA5-treated caps did not elongate significantly, they appeared “lumpier” than the smooth spherical structures formed in the absence of activin, suggesting that mAb AA5 did not completely block all morphogenetic movement. A different mAb that inhibits C-cadherin–mediated adhesion, 6B6, did not block elongation to the same extent as mAb AA5, even though it did affect the morphology of the elongated caps. Thus, preventing the activin-induced decrease in C-cadherin–mediated adhesion significantly inhibited the morphogenetic elongation of the tissue, indicating that the change in C-cadherin–mediated adhesion is required for the process.

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

Show MeSH
Related in: MedlinePlus