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Fig. 4. Injection of morpholino antisense oligonucleotides (MOs) directed against the translational initiation site of ß-catenin-2 can phenocopy the ichabod mutation, whereas MOs against ß-catenin-1 have no ventralizing effect. (A-C) Effect of injection of MO1 into wild-type embryos. Injection of 1 mM MO1 often results in a slight necrosis in the head (B) and 3 mM MO1 causes more severe necrosis and bent shortened tails (C), but in neither case were the embryos ventralized. A wild-type embryo at the same stage is shown for comparison (A). (D-G) Effect of injection of MO2 into wild-type embryos. Examples of Class 2 (E) and Class 1 (F) embryos obtained by injecting wild-type embryos with 3 mM MO2 are compared with a wild-type embryo (D) and an embryo injected with 3 mM MO2^mis (G). (H) The effects of injection of 3 mM MO2 can be rescued by co-injection of ß-catenin-2^* RNA (ß-catenin-2 RNA with an altered ribosome binding region that will not bind to MO2). Injection of 3 mM MO2 alone yielded a distribution of ventralized phenotypes (red bars). Injection of ß-catenin-2^* RNA alone had no ventralizing effect (compare green and yellow bars). Co-injection of MO2 and the RNA yielded mostly wild-type-appearing embryos, with only a few embryos exhibiting weak ventralization (blue bars). In this experiment, we classified non-ventralized embryos into two classes, wild-type and C5, with C5 embryos exhibiting kinky notochords. (I) Injection of MO2 into ichabod embryos shifted the phenotypic distribution to more-severe ventralized classes.

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