Study on b-Catenins mechanisms of regulation in zebrafish blastula embryo

Background: â-catenin is a central component of the cadherin cell adhesion complex but also it plays an essential role in the canonical-Wingless/Wnt signaling pathway. In vertebrates, one of the initial steps for the establishment of the correct dorso-ventral (D/V) pattern in the embryo is the cytoplasmic accumulation followed by nuclear localization of â-catenin in the cells of the prospective dorsal side of the embryo. In zebrafish there are two â-catenins, 92,7% identical. The mutant fish line Ichabod (ich), with a mutation in the region of the â-catenin2 promoter that causes a decrease in the maternal accumulation of â-catenin2 protein in the embryos, fail to nuclear localize â- catenins and to form a dorsal organizer, so the embryos become ventralized. Aims: Taking advantage of the zebrafish model and in particular of this fish line, we investigated the regulation of â-catenins nucleus-cytoplasm translocation and in particular why in ich â-catenin1 cannot compensate for the loss of â-catenin2. Materials and Methods: We analyzed by real-time PCR the levels of six genes involved in the canonical Wnt pathway: axin1 and axin2, pygopus1 and pygopus2, bcl9 and bcl9-2. Results: Unexpectedly, they are all up-regulated in ich embryos before and after mid-blastula-transition (MBT). Thus, ich embryos may have an overactive destruction complex, resulting in an increased degradation of â- catenin1. This is consistent with our finding that microinjection of a dominant negative Axin2 (it destroys the degradation complex) in ich embryo partially rescue ich phenotype. Conclusions: Our results confirm in vivo, previous in vitro work showing that the two zebrafish â-catenins C-terminal domain are important for the stability of the protein, probably because shielding it from the â-catenin destruction complex. This, results in higher stability of â-Catenin2 than â-Catenin1. These data are the first in vivo indication that differences in the â-catenins CTD result in different stability of these proteins