S2CS4, Fig. of the canonical Wnt pathway to the dorsal marginal blastomeres by defining the domain name where the Wnt8a activity gradient is usually above the threshold value necessary for triggering the canonical -catenin pathway. In summary, this study establishes that this zebrafish maternal dorsal determinant, Wnt8a, is required to localize the primary dorsal center, and that the extent of this domain is defined by the activity of two maternally provided Wnt antagonists, Sfrp1a and Frzb. mutation results in embryos with severe anterior and dorsal defects (3). This mutation exhibits variable expression with a fraction of embryos completely radialized and lacking in nuclear localization of -catenin at the dorsal margin in the high and sphere stages (3, 4). Complete radialization is also Raddeanin A observed after ablation of the vegetal part of the yolk cell during the first 20 min of development (5), a condition that removes maternal Raddeanin A dorsal determinants present at the vegetal pole of the egg. Inhibition of microtubule-dependent Raddeanin A transport of these determinants (6C8) results in similar phenotypes. This clearly establishes that the maternal Wnt/-catenin signaling pathway is activated by dorsal determinants transported from the vegetal pole to the future dorsal margin by a microtubule-dependent mechanism. In amphibians, the dorsal determinants were initially thought to correspond to intracellular proteins transducing the signal from the canonical Wnt/-catenin signaling pathway (9). However, this pathway has now been shown to be activated extracellularly Rabbit Polyclonal to IL18R in a process that requires Wnt11, Wnt5a, and FRL1 (10). Further studies revealed that Wnt5a and Wnt11 physically interact with each other to activate both canonical and noncanonical Wnt signaling required for dorsal axis formation (11). O-sulfation of specific tyrosine residues was found to be necessary for the interaction of Wnt11 with Wnt5a and for enhanced canonical signaling activity (12). In zebrafish, the identity of the dorsal determinant has been under investigation for a number of years, but it has not been identified yet. In this study, we show that Wnt8a (13), a Wnt ligand known to activate the canonical pathway, is the dorsal determinant in zebrafish. In addition, we establish that two maternally provided Wnt inhibitors, Sfrp1a (14) and Frzb (15), are essential to limit the spatial extent of the maternal Wnt/-catenin signaling pathway, restricting the nuclear accumulation of -catenin to the dorsalmost cells. Results and Discussion We initially hypothesized that the dorsal determinant in zebrafish is a Wnt ligand, on the basis of analogy with the mechanism described in and and (19), transcripts of this gene are only observed in blastomeres in zebrafish (Fig. S1). We found that Wnt8a is the sole Wnt gene for which transcripts accumulate at the vegetal pole of oocytes and of early zebrafish embryos (Fig. S1). In primary oocytes, strong accumulation of Wnt8a mRNA is observed in the Balbiani body (Fig. 1and indicate the limits of Wnt8a mRNA localization in the cortical cytoplasm. After fertilization, during early cleavage stages, Wnt8a transcripts are asymmetrically localized in the cortical cytoplasm on one side of the yolk cell and appear to move progressively to a more animal position (Fig. 1 and tests. value of <0.05 was considered statistically significant. Activation of the maternal -catenin pathway by Wnts, whose mRNAs are maternally supplied to the egg, appears specific to Wnt8a. The other canonical Wnts that display strong maternal expression (Wnt2, Wnt9b, and Wnt10b), and all noncanonical Wnts (Wnt4a, Wnt4b, Wnt5a, Wnt5b, Wnt11, and Wnt11r), are unable to induce dharma expression at the animal pole, even with injection of 20 times more mRNA than was used for Wnt8a (Fig. S3). Similarly, coinjection into one animal pole blastomere at the 64-cell stage of mRNAs coding for the two Wnts, Wnt11 and Wnt5a, known to be responsible for the activation of the maternal -catenin signaling pathway in amphibians (10C12), fails to induce dharma expression at the animal pole (Fig. S3and and and and mutant phenotype, are likely to be defective in the initial induction of the maternal Wnt/-catenin signaling pathway. Injection of Wnt8a efficiently rescued these ventralization phenotypes with a complete disappearance of radialized embryos and a statistically significant reduction in the number of embryos that are strongly ventralized (Fig. 2point to the accumulation of -catenin in blastomeres and yolk syncytial layer nuclei, Raddeanin A respectively. (and and and and and dominant-negative X-Wnt8 (33) and 500 pg of.
Categories