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Figure 2. Expression of α-catenin–GFP fusion proteins in Xenopus embryos and the localization of the β-catenin binding site. (A) Expression of proteins detected by anti-GFP. (B) After immunoprecipitations with anti–β-catenin, associated α-catenin was detected using anti-GFP. In both A and B, the numbering is as follows: lane 1, GFP injected; lane 2, uninjected; lane 3, αNcatNtermGFP + GFP; lane 4, αNcatNtermGFP + ΔArm; lane 5, GFPαNcatCterm; lane 6, GFPαNcat; lane 7, GFPαNcatNterm. Note that GFPαNcatCterm, the protein lacking the NH2 terminus of αN-catenin, does not bind to β-catenin (B, lane 5). Results show that the presence of GFP does not prevent α-catenin binding to β-catenin, provided that the NH2-terminal binding domain is present. In addition, coexpression of ΔArm does not affect expression levels. In each case, 1.5 ng total RNA was injected into one blastomere at the two-cell stage.
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Figure 3. Mutants lacking the COOH terminus of αN-catenin result in severe gastrulation defects. (A) GFPαNcat-injected embryos develop normally. (B) GFPαNcatCterm-expressing embryos develop normally. (C and D) Both αNcatNtermGFP- and GFPαNcatNterm-injected mRNAs induce defects beginning at stage 10, here shown by rips (arrows) occurring at the dorsal lips of the embryos. Embryos are shown with the ventral side toward the top. mRNA was injected into the animal pole of one cell of two-cell embryos.
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Figure 4. ÎArm binds α-cateninâ GFP fusion proteins. The 9E10 monoclonal antibody that recognizes the myc tag on ÎArm was used to immunoprecipitate, and anti-GFP was used with subsequent immunoblotting to detect α-cateninâGFP 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 + ÎArm; second lane, αNcatNtermGFP + ÎArm; third lane, uninjected.
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Figure 5. ÎArm and GFPαNcat rescue the defects caused by dominant-negative α-catenin mutants. (A, B, and C) αNcatNtermGFP + GFP, ÎArm, or GFPαNcat, respectively. (DâF) GFPαNcatNterm + GFP, ÎArm, or GFPαNcat. In all cases, 0.3 ng of αNcatNtermGFP or GFPαNcatNterm mRNA was coinjected with 1.2 ng of the second mRNA. Mutants in A and D do not develop further than gastrulation. Rescued mutants gastrulate (B and C, and E and F) and continue to develop normally. mRNA was injected into the animal pole of one cell of two-cell embryos.
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Figure 6. Histology of gastrulating embryos expressing α-cateninâ GFP fusion proteins. Stage 10â11 embryos were fixed in MEMFA and imbedded in paraplast (A and C) or plastic (B, D, E, and F) for sectioning. Arrowheads point to the blastocoel (bc). The abbreviation dl denotes the presumptive dorsal side of the embryos. (A) An uninjected normal embryo. (B) An embryo injected with GFPαNcatCterm mRNA, (C) αNcatNtermGFP, and (D) GFPαNcatNterm. Embryos expressing αNcatNtermGFP completely lack the blastocoel and are disorganized compared to normal embryos. Also noticeable is the lack of integrity of the ectodermal layers in the two mutants. Higher magnification emphasizes the disorganization of cells in an embryo expressing αNcatNtermGFP compared to a normal embryo expressing GFPαNcatCterm. mRNA was injected into the animal pole of both cells of two cell embryos. Bars: (AâD) 200 μm; (E and F) 100 μm.
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Figure 7. Redistribution of GFPαNcatNterm and αNcatNtermGFP from the glycoprotein fraction to the soluble fraction by ÎArm. (A) Soluble fractions immunoblotted with the anti-GFP polyclonal antibody. (B) Glycoprotein fractions immunoblotted with the anti-GFP polyclonal antibody. Lanes in both cases are 1, uninjected; 2, GFP injected; 3, GFPαNcatCterm injected; 4, αNcatNtermGFP + GFP; 5, αNcatNtermGFP + ÎArm; 6, GFPαNcatNterm + GFP; 7, GFPαNcatNterm + ÎArm; 8, GFPαNcat. Note the decrease in B between lanes 4 and 5 and lanes 6 and 7, implying that ÎArm acts by binding to the α-catenin mutants, keeping them from binding to the cadherin complex. When coinjections were done, 0.3 ng of the first mRNA was coninjected with 1.2 ng of the second mRNA.
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Figure 8. Levels of endogenous Xenopus α-catenin bound to cadherins decrease in embryos expressing the dominant-negative mutants αNcatNtermGFP or GFPαNcatNterm. Embryos were lysed in NP-40 buffer and immunoprecipitated using the monoclonal antibody to C-cadherin 6B6. The immunoprecipitates were immunoblotted with the polyclonal antiâαN-catenin antibody (CME). The lanes correspond to embryos injected with mRNA for 1, uninjected; 2, GFP; 3, αNcatNtermGFP; 4, GFPαNcatNterm; 5, uninjected. Lane 5 represents uninjected embryos subjected to immunoprecipitation with an anti-myc epitope antibody (9E10) as a negative control. Embryos were injected into both cells at the two-cell stage to obtain protein expression in the highest possible number of cells. Notice that the levels of endogenous α-catenin decrease significantly in lanes 3 and 4 compared to lanes 1 and 2.
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Figure 9. Animal cap assays reveal the dissociated nature of blastomeres expressing dominant-negative α-catenin. (A) Animal caps in 1à MMR normally heal and involute after 1 h as shown in GFPαNcat-injected embryos. (B) αNcatNtermGFP-expressing animal caps fail to involute, and dissociated blastomeres disperse after 1 h. (C) Dissociated GFP-expressing animal caps reaggregate in 2 mM Ca2+, whereas in D animal caps expressing αNcatNtermGFP do not. mRNA was injected into the animal pole of both cells of two-cell embryos.
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Figure 10. Coexpression of α-catenin diminishes the dorsalizing effects of Xwnt-8 and β-catenin. (A) Noninjected normal tadpoles. (B) Embryos expressing GFP + GFPαNcat. (C) Embryos expressing Xwnt-8 + GFP. (D) Xwnt-8 + GFPαNcat. (E) β-catenin + GFPαNcatCterm. (F) β-catenin + GFPαNcat. Embryos coexpressing α-catenin with Xwnt-8 develop longer trunks and are less dorsalized than embryos coexpressing GFP. Embryos ventrally expressing GFPαNcat + GFP develop normally (B). GFPαNcat antagonizes the induction of a secondary dorsoanterior axis by β-catenin (F). In B–D, embryos were coinjected with 0.125 ng of the first mRNA and 3 ng of the second. In E and F, 0.2 ng of β-catenin mRNA was coinjected with 3.0 ng of the αN-catenin constructs. One ventral blastomere was injected in each four-cell embryo. 1-mm glass beads hold the embryos upright.
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Figure 11. Full-length α-catenin can antagonize the endogenous induction of a dorsoanterior axis when expressed in dorsal blastomeres. 3.0 ng of either GFPαNcatCterm, which does not bind to β-catenin, or GFPαNcat, the full-length construct, were injected into both dorsal blastomeres of four-cell embryos. Embryos expressing GFPαNcat often develop the ventralized phenotypes illustrated here (B and D), with no anterior head structures present. Embryos expressing high levels of GFPαNcatCterm develop normally (A and C). Two stages are shown.
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Figure 12. (A) α-Catenin inhibits the induction of the homeobox-containing gene, Siamois, by full-length β-catenin or Xwnt-8 in animal caps. One cell of two-cell embryos was injected in the animal hemisphere, and animal cap explants were taken at stage 9 and allowed to develop to stage 10.5, when total RNA was prepared. In lanes 2–6, coinjections were with 0.125 ng of the first mRNA and 3.0 ng of the second. Lane 1, uninjected whole embryos; lane 2, full-length β-catenin + GFP; lane 3, full-length β-catenin + αNcatNtermGFP; lane 4, full-length β-catenin + GFPαNcat; lane 5, GFP + αNcatNtermGFP; lane 6, GFP + GFPαNcat; lane 7, 0.125 ng Xwnt-8 + 3.0 ng GFPαNcatCterm; lane 8, 0.125 ng Xwnt-8 + 3.0 ng GFPαNcat; lane 9, 0.125 ng Xwnt-8 + 1.5 ng GFP; lane 10, 0.125 ng Xwnt-8 + 3 ng GFP; lane 11, 0.125 ng Xwnt-8 + 0.75 ng GFPαNcat; lane 12, 0.125 ng Xwnt-8 + 1.5 ng GFPαNcat; lane 13, 0.125 ng Xwnt-8 + 3 ng GFPαNcat; 14, 0.125 ng GFP + 3 ng GFPαNcat. The ubiquitously expressed elongation factor 1α (EF1α) was included as a loading control. RNAse protection assays show that either full-length αN-catenin (GFPαNcat) or the dominant-negative αN-catenin (αNcatNtermGFP) decrease the levels of Siamois mRNA induced in animal caps by β-catenin or Xwnt-8. Moreoever, increasing concentrations of GFPαNcat result in proportionate decreases in Siamois induction by Xwnt-8. (B) α-Catenin inhibits the endogenous expression of Siamois when injected into dorsal blastomeres at the four-cell stage. Both dorsal blastomeres were injected with mRNA, and embryos were extracted for total RNA at stage 10.5. Lane 1, 1.5 ng GFPαNcat; lane 2, 3.0 ng GFPαNcat; lane 3, 3.0 ng GFPαNcatCterm; lane 4, uninjected embryos. Embryos injected with GFPαNcat mRNA (which develop into ventralized embryos) express lower levels of Siamois than normal embryos or embryos injected with GFPαNcatCterm (which develop normally).
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