FIG. 1. The effectiveness of beta-catenin MO in causing ventralization and reducing beta-catenin protein levels. 6 ng of morpholino oligo (Mo) was injected into two cells (3 ng each) at the two-cell stage. This caused a ventralized phenotype compared to uninjected (Uninj) controls. (B) Real-time RT PCR analysis of sibling embryos of those in A, for molecular markers of dorsal development siamois, Xnr3, and chordin and the ventral marker Xwnt 8. The level of expression of these markers is compared in control, MO iniected , and MO + beta-catenin mRNA at the early gastrula stage (see Materials and Methods for details). While dorsal markers were reduced by MO injection, the ventral marker -was increased. When MO was injected together with beta-catenin mRNA, the ventralized phenotype was reversed and dorsal markers were hyperexpressed. (C) When MO was injected together with 500 pg beta-catenin mRNA, the ventralized phenotype was reversed and dorsalized embryos resulted (Mo + RNA). (D) Northern blotting shows that the morpholino oligo injected at 6 and 12 ng did not degrade beta-catenin mRNA at the blastula or neurula stages. A probe for plakoglobin mRNA was used as a loading control. (E) The degree of depletion of beta-catenin protein at early gastruta (stage 10) and tailbud (stage 24) stages is compared in a Western blot. When the oligo was injected into both cells of the two-cell stage embryo, or into the two dorsal cells of the four~cell stage embryo, it caused a significant reduction in total beta-catenin protein at the early gastrula stage but not at the tailbud stage. ~-Tubulin is used as a loading control, cs, cell stage; dors, dorsal injection. IF) MO caused a greater depletion of beta-catenin protein at the neurula stage than at the blastula stage and reduced protein levels in both membrane and cytosolic fractions. Sibling embryos of those shown in A were Western blotted in pellet and supernatant fractions at the stages shown. Significant reduction of protein levels occurred in both fractions compared to the uninjected control.
Image published in: Heasman J et al. (2000)
Copyright © 2000. Image reproduced with permission of the Publisher, Elsevier B. V.
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