The Wnt signaling network has critical roles in development and disease. Simplified, this complex network has two distinct outputs: the Wnt/β-catenin module activates the phosphoprotein Dishevelled (Dvl) and leads to transcriptional activation while the Wnt/Planar Cell Polarity (PCP) module activates Dvl and leads to calcium release and directed cell movement. Wnt/β-catenin and Wnt/PCP share signaling components like Frizzled receptors, Dvl, and Naked (Nkd). It is an open question how converging Wnt signals diverge into separate outcomes. In this thesis, I used molecular techniques, functional studies in the zebrafish, and biochemical approaches to determine the role of Nkd in Wnt signaling.
Nkd contains and EF-hand, a putative calcium binding domain, and is known to antagonize Wnt/β-catenin and disrupt Wnt/PCP signaling. We utilized a tissue that requires both Wnt/β-catenin and Wnt/PCP signaling to properly pattern the left/right axes of the embryo; the dorsal forerunner cells (DFCs). The DFCs exhibit aperiodic calcium release as they migrate to form the Kupffer's Vesicle (KV), the organ of asymmetry. Calcium inhibition in the DFCs disrupts their migration, alters KV formation, and disrupts left/right patterning. Nkd is enriched in the DFCs during migration and KV formation and endogenous Nkd knockdown in the DFCs produces the same phenotypes as calcium inhibition, making Nkd a candidate molecule for directing converging Wnt signals to distinct outcomes.
To assess the role of the EF-hand in Nkd function, I created point mutations predicted to disrupt EF-hand affinity for calcium. Through functional studies in zebrafish embryos, I determined that Nkd EF-hand is necessary for Nkd function in Wnt/PCP signaling, but dispensable for Wnt/β-catenin signaling. Although Nkd has not been shown to bind calcium, our functional data with the Nkd EF-hand point mutant provides compelling evidence for a role for calcium in Nkd function in directing Wnt signaling output.
EF-hand affinity for calcium is influenced by binding partners, and since Nkd binds to Dvl in the Dvl PDZ domain, we screened the domain for a region rich in amino acids that facilitate ion binding. We identified a 12-amino acid sequence in the Dvl PDZ domain with potential to create a negatively charged pocket to help coordinate calcium binding. We expressed the Nkd EF-hand (EFX) Dvl basic domain and PDZ domain (bPDZ). The purified EFX and bPDZ constructs were used to investigate the interaction between Nkd, Dvl, and calcium. I show, by circular dichroism, that the Nkd/Dvl complex undergoes a calcium-induced change in secondary structure. This reveals the mechanism by which Nkd directs Dvl from the default Wnt/β-catenin signaling module to the Wnt/PCP module in response to calcium.