RNA Helicase DDX3 Is a Regulatory Subunit of Casein Kinase 1 in Wnt–β-Catenin Signaling
Reinoud E. A. de Groot2,
Hendrik C. Korswagen2, and
1 Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
2 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Netherlands.
3 Institute of Molecular Biology, D-55128 Mainz, Germany.
View larger version (50K):
[in this window]
[in a new window]
Fig. 1. DDX3 is required for Wnt–β-catenin signaling in mammalian cells and during a-p neural patterning in Xenopus. (A) Wnt luciferase reporter assay in HEK293T cells stimulated with Wnt3a-conditioned medium or by transfection with the indicated constructs, in the presence of the indicated siRNAs. RLA, relative luciferase activity. Error bars indicate SDs; n = 3, biological triplicates of one representative assay. (B) Western blot analysis of endogenous β-catenin from cytosolic and nuclear fractions of HEK293T cells stimulated with control or Wnt3a-conditioned medium in the presence of the indicated siRNAs. (C) Xenopus axis duplication assay by injection of the indicated mRNAs into the ventral blastomeres of four-cell-stage embryos. (D) Quantitative polymerase chain reaction (QPCR) analysis of siamois using VMZ explants from Xenopus embryos injected with the indicated mRNAs. Explants were excised and analyzed from gastrula-stage embryos. Error bars indicate SDs; n = 2 assays. VMZ, ventral marginal zone; sia, siamois. (E) Tadpole-stage Xenopus embryos that were injected at the two-cell stage in the animal hemisphere with DDX3 or LRP6 antisense Mo oligonucleotides in the absence or presence of human DDX3 mRNA (WT, wild type) as indicated. (F) Whole-mount in situ hybridization of neurula-stage embryos injected at four-cell stage in animal blastomeres with the indicated Mo plus β-galactosidase mRNA lineage tracer (red; arrow marks injected side).|