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Fig. 2. In-vivo measurements of ß-catenin-GFP half-life. (A-C) Measurement of ß-catenin-GFP half-life at the 16-cell stage. Confocal projections (vegetal views) of an embryo expressing Xl-wt-ß-catenin-GFP. Emetine was added immediately after the third cleavage division (A), and after 30 minutes in the presence of the inhibitor the embryo had completed fourth cleavage (B). Loss of ß-catenin-GFP was apparent in animal blastomeres during the ensuing 30 minutes (arrows, B,C). (D) Representative data from a single embryo, showing decay of GFP fluorescence in specific cell lineages as measured by 4-D confocal microscopy. 0 on the x-axis corresponds to 30 minutes after addition of emetine. (E) Control experiment measuring ³⁵S-methionine incorporation after 25 minutes' exposure to varying concentrations of emetine. 100 µM emetine blocked 90% of new protein synthesis. (F) Control for GFP photobleaching. Fertilized eggs were injected with mRNA encoding GFP and allowed to develop to the 8-cell stage. Embryos were treated with 100 µM emetine for 30 minutes and then imaged with 4-D confocal microscopy under conditions identical to those shown in Fig. 1. These representative data from a single embryo show that GFP fluorescence (mean fluorescent pixel intensity measured over the entire embryo) remained constant over the period of the experiment. 0 on the x-axis corresponds to 30 minutes after addition of emetine. (G) Decay in GFP fluorescence is dependent on ß-catenin phosphorylation. Fertilized eggs were injected with Xl-pt-ß-catenin-GFP mRNA. At the 8-cell stage, embryos were treated with 100 µM emetine for 30 minutes and then imaged with 4-D confocal microscopy under conditions identical to those shown in Fig. 1. These representative data from a single embryo show that GFP fluorescence (mean fluorescent pixel intensity measured over the entire embryo) remained constant over the period of the experiment. (H) Summary of Xl-wt-ß-catenin-GFP half-life measurements. Half-life was measured in different cell lineages of cleavage stage embryos and data were pooled from 8-, 16- and 64-cell stage embryos. On average, within any individual embryo, the half-life of ß-catenin-GFP in the vegetal-most blastomeres (the micromere territory) was more than 8-fold greater than in the animal blastomeres (the mesomere territory).

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