Characterization of a novel component of Wnt signaling pathway using zebrafish as a model organism.

Abstract

Wnt signaling plays important role in many aspects of embryogenesis such as cell proliferation, cell fate specification, cell polarity and organogenesis(Clevers 2006, van Amerongen and Nusse 2009). Wnt ligands have been shown to activate several intra-cellular signaling cascades, including the canonical or Wnt/-catenin dependent pathway and the non-canonical or -catenin independent pathway. Dishevelled (Dvl) occupies a key position at crossroads of all branches of Wnt signaling cascade. To understand, how Dishevelled (Dvl) may channel signaling into the downstream branches, we sought to identify novel effectors for Dishevelled (Dvl) using a yeast-two hybrid screen. In this study, we used the PDZ domain of Dishevelled (Dvl) as a bait and from this screen, we identified a new binding protein of Dishevelled (Dvl)-termed as Custos. To characterize the functional role of Custos in Wnt signaling pathway, we used mammalian cell culture and zebrafish as a model vertebrate organism. We confirmed the interaction between Custos and Dvl using co-immunoprecipation and GST pull-down. Custos also interacted with -catenin in vivo and this interaction was positively regulated by Wnt stimulation. Immunofluorescence experiments in mammalian cells showed that Custos co-localizes with the nuclear envelope marker, lamin and inhibits translocation of -catenin to the nucleus. In zebrafish embryos, Custos is a maternal gene and expressed throughout development. Spatial in situ hybridization studies showed that Custos was expressed in the dorsal region of the embryo at early stages and in the nervous system in zebrafish at 24hpf. To delineate the biological role of Custos during embryogenesis, we conducted a gain of function and loss of function studies. Overexpression of exogenous Custos and morpholino knockdown of Custos revealed that Custos is critical for embryonic patterning. Knockout of Custos in zebrafish revealed that Custos delays embryonic development and exhibits defects in pigmentation suggesting a plausible role in neural crest development. Taken together, our studies demonstrate that Custos is a novel component of canonical Wnt signaling and required for -catenin translocation into the nucleus and important for embryonic patterning.