Dishevelled (Dvl) is a cytoplasmic protein involved in the Wnt-Frizzled signaling cascade, which has also been shown to interact with the cytoskeleton in part through inhibition of glycogen synthase kinase 3beta (GSK3beta). Using mouse neuroblastoma 2A (N2A) cells as a model system, we have found that overexpression of Dvl promotes the outgrowth of neurite-like processes, and leads to the induction of a striking, bipolar morphologic phenotype during neuronal differentiation. In contrast, suppression of Dvl expression using isoform-specific siRNAs led to an inhibition of neurite outgrowth in these cells. In order to further elucidate the mechanism(s) responsible for this effect, we overexpressed several mutant forms of Dvl in the N2A cells, including deletions in each of the three major functional subdomains of the protein (DeltaDIX, DeltaPDZ, DeltaDEP) and point mutations in the two well-defined interaction motifs within the DIX domain (the actin-binding and vesicle-association elements; K58A and K68A/E69A, respectively). These experiments revealed that the DIX domain (and its vesicle-binding subregion) was essential for Dvl's effect on neurite extension and morphogenesis in N2A cells. In contrast, direct overexpression of a degradation-resistant form of beta-catenin (S37A), or a dominant negative GSK3beta mutant (K85R), had no effect on neurite outgrowth or morphology in neuronally differentiating N2A cells; exposure of cells to a pharmacologic inhibitor of GSK3beta (lithium) also had no effect. Taken together, these results suggest that Dvl induces cytoskeletal and morphologic rearrangements in neuronal differentiating N2A cells through a mechanism that cannot be attributed exclusively to modulation of GSK3beta or beta-catenin activity, but which does depend upon a DIX-domain/vesicle-association-dependent signaling pathway.