Study on the regulation of Dishevelled for Wnt signaling during Xenopus embryonic development

Abstract

During vertebrate embryogenesis, diverse signaling pathways are involved to accomplish the precise development. One of the most crucial signaling cascade responsible for the proper embryogenesis is the Wnt signaling pathway. Signaling by Wnt proteins control numerous developmental processes including dorso-ventral body axis formation, antero-posterior patterning, mesoderm formation, tissue morphogenesis and organogenesis. In addition, Wnt signaling is required for adult tissue maintenance and dysregulation of Wnt itself or its downstream signaling components causes human degenerative diseases, birth defects and cancer. Wnt signaling pathways are branched off to the β-catenin-dependent canonical pathway and the β-catenin-independent non-canonical pathway. Although each pathway confers different developmental processes, Dishevelled (Dvl) serves as a scaffold protein that plays central roles in both Wnt signaling pathways by transmitting Wnt stimulation from receptors to downstream effector proteins. Despite of recent characterization of Dvl interacting partners and their regulatory functions, 1. The mechanisms by which Dvl differentially activates each Wnt pathways and 2. The mechanisms by which stability of Dvl protein is regulated are still not fully understood. In this study, we examined the involvement of Dvl-cholesterol interaction for the differential activation of Wnt pathways and function of E3 ubiquitin ligase March2 for the regulation of Dvl stability during Xenopus embryogenesis.

1. Cholesterol Binding is a Determinant of Canonical Wnt Signaling Activity of Dishevelled Dishevelled (Dvl) is a scaffold protein that serves as the branch point of canonical Wnt and PCP signaling but it remains unclear how Dvl differentially directs the two pathways. Here, we show that Dvl is a novel cholesterol binding protein and this Dvl-cholesterol binding on the plasma membrane selectively drives the progression of canonical Wnt signaling over non-canonical/PCP signaling. In Xenopus embryos, cholesterol binding of Dvl is required for canonical Wnt signaling activities but not for PCP signaling activities. Furthermore, confocal and single molecule imaging using culture cells shows that cholesterol is essential for sustained association of Dvl with the Frizzled receptor and other components of canonical Wnt signaling in the plasma membrane and endocytic vesicles in response to a canonical Wnt ligand, but not for the formation of the PCP signaling complex. Collectively, our results suggest a new role of cholesterol binding of Dvl in Wnt signaling during Xenopus embryogenesis and a potential link between cellular cholesterol levels and canonical Wnt signaling activities.

2. March2 regulates Wnt signaling pathways by targeting Dishevelled for degradation during Xenopus development Here we identify March2, a RING domain-containing ubiquitin ligase, targeting Dvl protein and show that it regulates canonical Wnt signaling in Xenopus development. First, we intriguingly found that March2 depletion by microinjection of morpholino oligonucleotide caused the defects in head formation. We also found that these defective phenotypes were caused by Wnt antagonistic function of March2. Reporter assay, western blot analysis for phosphorylation of LRP6 and activation of b-catenin, RT-PCR and axis duplication assay reveled that March2 inhibits canonical Wnt activities. Furthermore, head defective phenotypes from March2 morphants could be rescued by coexpression of Axin1, a negative regulator of canonical Wnt signaling. We also found that March2 interacts with DEP domain of Dvl and mediates its poly-ubiquitination. Taken together, these results suggest that March2 negatively regulates Wnt signaling by targeting Dvl protein for ubiquitin-mediated degradation for precise head development.