Regulation of the differential nuclear localization of beta-catenin in the early sea urchin embryo

Differential nuclear accumulation of β-catenin along one axis of the early embryo is required for mesoderm and/or endoderm formation in deuterostomes and is important in the development of early signaling centers. The mechanisms underlying this critical developmental step, however, are unknown.; mRNA encoding Xenopus β-catenin-GFP was microinjected into fertilized eggs and the dynamics of β-catenin localization were observed in vivo using 3-D time-lapse confocal microscopy. β-catenin-GFP initially accumulated in the nuclei of all cells. Beginning at the 32-cell stage, I observed a dynamic loss of β-catenin-GFP from the animal two-thirds of the embryo. Embryos injected with hyperstable β-catenin-GFP mRNA maintained nuclear β-catenin in all cells, supporting the hypothesis that GSK-3β phosphorylation sites are important for β-catenin degradation. Embryos co-expressing kinase-dead GSK-3β and β-catenin-GFP also retained nuclear β-catenin in all cells. The half-life of β-catenin-GFP was quantitated by measuring changes in fluorescent intensity in tiers of blastomeres along the animal-vegetal axis. I found, on average, an 8-fold difference in β-catenin stability when comparing the animal and vegetal-most cells. Together, these data provide the first experimental evidence for a gradient of β-catenin stability along an axis and support the view that degradation of β-catenin occurs via a GSK-3β-dependent mechanism.; Regulation of nuclear β-catenin stability is likely to be dependent on the differential localization, stability, or activity of other β-catenin pathway components. I found that Dishevelled-GFP is asymmetrically localized in the vegetal cortex prior to the first cleavage and is maintained in the micromeres throughout early development. Dishevelled, therefore, is in the right place at the right time to be involved in β-catenin stabilization. In support of this view, overexpression of the Dishevelled DIX domain alone acts as a dominant negative and blocks nuclearization of β-catenin. A detailed structure/function analysis of the Dishevelled sequence was performed to determine which domains/regions are involved in its asymmetric localization. I discovered that the region between PDZ and DEP is required for targeting Dishevelled-GFP to the vegetal cortex, while all other domains are dispensable. Furthermore, experiments using cytoskeletal inhibitors indicated that microfilaments, but not microtubules, are necessary for Dishevelled to localize to the vegetal cortex.