Modulation of the β-Catenin Signaling Pathway by the Dishevelled-Associated Protein Hipk1

BACKGROUND:Wnts are evolutionarily conserved ligands that signal through beta-catenin-dependent and beta-catenin-independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. METHODOLOGY:We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/beta-catenin target gene activation, as demonstrated by beta-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. CONCLUSIONS:These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/beta-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation

Functional conservation of the Wnt signaling pathway revealed by ectopic expression of Drosophila dishevelled in Xenopus.

Wnt genes encode secreted growth factors that exhibit potent effects on both embryonic and postembryonic development in vertebrates and invertebrates. Recently, the dishevelled (dsh), shaggy/zeste-white 3, and armadillo genes have been shown to participate in Wnt (wingless; wg) signaling in Drosophila. Vertebrate genes that have sequence similarities to all of these Drosophila genes have been identified. To determine whether these structurally conserved components of insect wg signaling represent a functionally conserved Wnt signaling pathway in vertebrates, we investigated the role of Drosophila dsh in Xenopus Wnt signaling. Xenopus embryos ectopically injected with Drosophila dsh mRNA developed duplicated axes similar to those seen in embryos injected with Wnt mRNAs. The involvement of dsh function in the Wnt signaling pathway in Xenopus was demonstrated using two assays which are specifically sensitive to Wnt signaling: synergistic induction of dorsal mesoderm with bFGF and the specific induction of a Wnt-responsive reporter gene. These findings support the notion that the intracellular response to the Wnt signal has been conserved during evolution to such an extent that its components may be interchanged between distantly related species

Functional conservation of the Wnt signaling pathway revealed by ectopic expression of Drosophila dishevelled in Xenopus.

Wnt genes encode secreted growth factors that exhibit potent effects on both embryonic and postembryonic development in vertebrates and invertebrates. Recently, the dishevelled (dsh), shaggy/zeste-white 3, and armadillo genes have been shown to participate in Wnt (wingless; wg) signaling in Drosophila. Vertebrate genes that have sequence similarities to all of these Drosophila genes have been identified. To determine whether these structurally conserved components of insect wg signaling represent a functionally conserved Wnt signaling pathway in vertebrates, we investigated the role of Drosophila dsh in Xenopus Wnt signaling. Xenopus embryos ectopically injected with Drosophila dsh mRNA developed duplicated axes similar to those seen in embryos injected with Wnt mRNAs. The involvement of dsh function in the Wnt signaling pathway in Xenopus was demonstrated using two assays which are specifically sensitive to Wnt signaling: synergistic induction of dorsal mesoderm with bFGF and the specific induction of a Wnt-responsive reporter gene. These findings support the notion that the intracellular response to the Wnt signal has been conserved during evolution to such an extent that its components may be interchanged between distantly related species