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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: To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin.Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation.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: To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin.Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation.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