Identification and Functional Characterization of Dishevelled-Interacting Proteins in the Sea Urchin Strongylocentrotus Purpuratus and the Cnidarian Nematostella Vectensis

Wnt signaling plays a central role in establishing anterior-posterior (AP) polarity in metazoan embryos. A key cytoplasmic component mediating Wnt signaling is the Disheveled (Dvl) protein, which is generally considered to be the central “hub” of the Wnt signaling pathway. In the sea urchin, Dvl is highly enriched and differentially post-translationally modified in a specialized vegetal cortical domain (VCD) of the egg, and the vegetal blastomeres that inherit the VCD during embryogenesis. Functional analysis has shown that localized Dvl activity mediates Wnt/beta-catenin signaling pathway in vegetal blastomeres, but the molecular basis of Dvl asymmetric localization and activation remain unresolved. Therefore, identification and functional characterization of proteins interacting with Dvl (DIPs) in the VCD will help us better understand how Dvl partners regulate Dvl activity and Wnt signaling. In this work, I identified several hundred potential Dvl-interacting-proteins (DIPs) from isolated egg cortices and 16-cell-stage micromeres by carrying out Dvl Co-Immunoprecipitation followed by mass-spectrometry. The functionality enrichment analysis revealed some common enriched GO terms for the identified DIP candidates identified from the cortex and the micromere, such as cytoskeletal proteins, translation initiation factor activity, binding, ribosome, ribosomal protein vesicle coat and the Wnt signaling pathway. The comparison of the functionality enrichment and protein complex analyses between the DIPs candidates identified from the cortex and the DIP candidates identified from the micromere indicates that Dvl activity is dynamically regulated between the egg cortex and the 16-cell stage micromeres. Casein Kinase 1 δ/ε (CK1δ/ε), one of our identified DIP candidates, is highly enriched and co-localized with Dvl at the vegetal pole of the sea urchin embryo. Downregulation of CK1δ/ε by overexpressing a dominant-negative form of CK1δ/ε resulted in the anteriorization of embryos and a severe downregulation of endomesodermal gene expression. Furthermore, overexpression of CK1δ/ε by injecting synthesized CK1δ/ε mRNA into fertilized eggs induced slight upregulation of endomesoderm genes. Intriguingly, we found that co-overexpressing CK1δ/ε and Dvl induces a more severe posteriorized phenotype than when overexpressing CK1δ/ε alone, suggesting that CK1δ/ε synergizes with Dvl to positively regulate Wnt signaling. This hypothesis was further supported by the observation that the expression levels of endomesoderm genes were significantly higher in CK1δ/ε and Dvl co-overexpressed embryos compared to expression levels of endomesoderm genes in embryos overexpressing Dvl or CK1δ/ε only. This work establishes CK1δ/ε as a critical regulator of Dvl activation and AP axis specification in sea urchin embryos. Functional studies for the other selected DIPs candidates in the sea urchin, including SpCK2α, SpYap and SpDDX5/17, suggested the important role of the protein network formed around Dvl and SpCK1δ/ε in regulating Wnt signaling and endomesoderm specification. Comparative studies for selected DIP candidates between the sea urchin and Nematostella vectensis provided critical insights into the mechanisms underlying the evolution of the body plan organization of metazoans