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Antagonism of cell adhesion by an alpha-catenin mutant, and of the Wnt-signaling pathway by alpha-catenin in Xenopus embryos.

Sehgal RN, Gumbiner BM, Reichardt LF - J. Cell Biol. (1997)

Bottom Line: Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation.We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8.This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Department of Biochemistry and Biophysics, and Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0724, USA.

ABSTRACT
In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based cytoskeleton. To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant of beta-catenin that lacks the internal armadillo repeats. We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular beta-catenin signaling pathway in vivo.

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Related in: MedlinePlus

ΔArm binds α-catenin– GFP fusion proteins. The 9E10  monoclonal antibody that recognizes the myc tag on ΔArm was  used to immunoprecipitate, and  anti-GFP was used with subsequent  immunoblotting to detect α-catenin–GFP fusion proteins in the precipitates. Embryos were coinjected  with 0.3 ng of the first mRNA and  1.2 ng of the second. First lane, GFP  + ΔArm; second lane, αNcatNtermGFP + ΔArm; third lane, uninjected.
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Figure 4: ΔArm binds α-catenin– GFP fusion proteins. The 9E10 monoclonal antibody that recognizes the myc tag on ΔArm was used to immunoprecipitate, and anti-GFP was used with subsequent immunoblotting to detect α-catenin–GFP fusion proteins in the precipitates. Embryos were coinjected with 0.3 ng of the first mRNA and 1.2 ng of the second. First lane, GFP + ΔArm; second lane, αNcatNtermGFP + ΔArm; third lane, uninjected.

Mentions: To ensure that the effects due to the mRNA injections were specific for α-catenin, we performed coinjection experiments. The first mRNA coinjected was a β-catenin construct lacking the internal armadillo repeats (ΔArm), which was described previously as ΔR in Kypta et al. (1996). This protein has been shown to retain binding to α-catenin but does not bind to cadherins or APC (Funayama et al., 1995). Fig. 4 shows that αNcatNtermGFP bound to immunoprecipitates of ΔArm in Xenopus extracts. Coexpression of ΔArm with either αNcatNtermGFP or GFPαNcatNterm resulted in a complete rescue of the phenotype, when the ratio of rescuing mRNA to αN-catenin truncation mRNA was 4:1 (Fig. 5, B and E, and Table II). Coinjecting the same amount of GFP mRNA as a control did not rescue the phenotype (Fig. 5, A and D, and Table II).


Antagonism of cell adhesion by an alpha-catenin mutant, and of the Wnt-signaling pathway by alpha-catenin in Xenopus embryos.

Sehgal RN, Gumbiner BM, Reichardt LF - J. Cell Biol. (1997)

ΔArm binds α-catenin– GFP fusion proteins. The 9E10  monoclonal antibody that recognizes the myc tag on ΔArm was  used to immunoprecipitate, and  anti-GFP was used with subsequent  immunoblotting to detect α-catenin–GFP fusion proteins in the precipitates. Embryos were coinjected  with 0.3 ng of the first mRNA and  1.2 ng of the second. First lane, GFP  + ΔArm; second lane, αNcatNtermGFP + ΔArm; third lane, uninjected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139960&req=5

Figure 4: ΔArm binds α-catenin– GFP fusion proteins. The 9E10 monoclonal antibody that recognizes the myc tag on ΔArm was used to immunoprecipitate, and anti-GFP was used with subsequent immunoblotting to detect α-catenin–GFP fusion proteins in the precipitates. Embryos were coinjected with 0.3 ng of the first mRNA and 1.2 ng of the second. First lane, GFP + ΔArm; second lane, αNcatNtermGFP + ΔArm; third lane, uninjected.
Mentions: To ensure that the effects due to the mRNA injections were specific for α-catenin, we performed coinjection experiments. The first mRNA coinjected was a β-catenin construct lacking the internal armadillo repeats (ΔArm), which was described previously as ΔR in Kypta et al. (1996). This protein has been shown to retain binding to α-catenin but does not bind to cadherins or APC (Funayama et al., 1995). Fig. 4 shows that αNcatNtermGFP bound to immunoprecipitates of ΔArm in Xenopus extracts. Coexpression of ΔArm with either αNcatNtermGFP or GFPαNcatNterm resulted in a complete rescue of the phenotype, when the ratio of rescuing mRNA to αN-catenin truncation mRNA was 4:1 (Fig. 5, B and E, and Table II). Coinjecting the same amount of GFP mRNA as a control did not rescue the phenotype (Fig. 5, A and D, and Table II).

Bottom Line: Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation.We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8.This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Department of Biochemistry and Biophysics, and Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0724, USA.

ABSTRACT
In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based cytoskeleton. To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant of beta-catenin that lacks the internal armadillo repeats. We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular beta-catenin signaling pathway in vivo.

Show MeSH
Related in: MedlinePlus