Search

Wnt proteins regulate diverse biological responses by initiating two general outcomes: β-catenin-dependent transcription and β-catenin-independent activation of signaling cascades, the latter including modulation of calcium and regulation of cytoskeletal dynamics (Planar Cell Polarity, PCP). It has been difficult to elucidate the mechanisms by which Wnt signals are directed to effect one or the other outcome due to shared signaling proteins between the β-catenin-dependent and -independent pathways, such as the Dishevelled binding protein Naked. While all Naked paralogs contain a putative calcium-binding domain, the EF-Hand, Drosophila Naked does not bind calcium. Here we find a lineage-specific evolutionary change within the Drosophila Naked EF-hand that is not shared with other insects or vertebrates. We demonstrate the necessary role of the EF-hand for Nkd localization changes in calcium fluxing cells and using in vivo assays, we identify a role for the zebrafish Naked EF-hand in PCP but not in β-catenin antagonism. In contrast, Drosophila-like Nkd does not function in PCP, but is a robust antagonist of Wnt/β-catenin signaling. This work reveals that the zebrafish Nkd1 EF-hand is essential to balance Wnt signaling inputs and modulate the appropriate outputs, while the Drosophila-like EF-Hand primarily functions in β-catenin signaling., (Copyright © 2017. Published by Elsevier Inc.)

Many aspects of animal development including fertilization as well as organ formation and function are dependent upon the dynamic release of calcium (Ca(2+)) ions. Although the controlled release and/or accumulation of Ca(2+) ions has been extensively studied, how the release dynamics produce a specific biological output in embryonic development is less clear. We will briefly summarize Ca(2+) sources, highlight data on endogenous Ca(2+) release in vertebrate embryos relevant to body plan formation and cell movement, and integrate pharmacological and molecular-genetic studies to lend insight into the signalling pathways involved. Finally, based on in vivo imaging in zebrafish genetic mutants, we will put forward the model that distinct Ca(2+) release dynamics lead to antagonism of the developmentally important Wnt/beta-catenin signalling pathway, while sustained Ca(2+) release modulates cell polarization or directed migration.

A zebrafish maternal effect mutation, in the gene hecate, results in embryos that have defects in the formation of dorsoanterior structures and altered calcium release. hecate mutant embryos lack nuclear accumulation of beta-catenin and have reduced expression of genes specific to the dorsal organizer. We found that hecate mutant embryos exhibit a nearly 10-fold increase in the frequency of intracellular Ca2+ transients normally present in the enveloping layer during the blastula stages. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in mutant embryos suggesting that Ca2+ transients are important in mediating dorsal gene expression. Inhibition of Ca2+ release also results in the expression of dorsal-specific genes in the enveloping layer in a beta-catenin-independent manner, which suggests an additional function for the Ca2+ transients in this cellular layer. The mutant phenotype can be reversed by the expression of factors that activate Wnt/beta-catenin signaling, suggesting that the Wnt/beta-catenin pathway, at least as activated by an exogenous Wnt ligand, is intact in hec mutant embryos. Our results are consistent with a role for the hecate gene in the regulation of Ca2+ release during the cleavage stages, which in turn influences dorsal gene expression in both marginal cells along the dorsoventral axis and in the enveloping layer.

The phosphoinositide (PI) cycle is an important signal transduction pathway that, upon activation, generates intracellular second messengers and leads to calcium release. To determine whether PI cycle-mediated intracellular calcium release is required for body plan formation, we systematically dissect PI cycle function in the zebrafish (Danio rerio). We inhibit PI cycle function at three different steps and deplete internal calcium stores, demonstrating an impact on endogenous calcium release and Wnt/beta-catenin signaling. Inhibition of endogenous calcium modulation induces hyperdorsalized phenotypes in a dose-dependent manner. Ectopic dorsal-signaling centers are generated in PI cycle-inhibited embryos as demonstrated by altered beta-catenin subcellular localization and ectopic expression of beta-catenin target genes. These results provide evidence that modulation of calcium release is critical for early embryonic patterning and acts by influencing the stabilization of beta-catenin protein.