Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The Dickkopf (Dkk) family is composed of four main members (Dkk1-4), which typically regulate Wnt/beta-catenin signaling. An exception is Dkk3, which does not affect Wnt/beta-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, headmesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.
Fig. 1 Expression of dkk3 during Xenopus laevis development. (A) Dkk homology tree. Percent protein sequence homology is indicated on the top. (B–F) Whole-mount in situ hybridization of dkk3 during Xenopus development. (B) Neurula stage (stage 17), (C) taibud stage, and (D) tadpole stage. Anterior is to the left. (E–F) Sagittal sections through the regions indicated in (D). (G) Real-time RT-PCR analysis of the indicated genes in Xenopus development. (H) RT-PCR of the indicated genes in dissected Xenopus stage 8 blastulae (D, dorsal; V, ventral explants). cg, cement gland; h, heart; n, notochord; ov, otic vesicle.
Fig. 2 Dkk3 is required for Xenopus axis formation. (A–J) One- to two-cell embryos were injected with two dkk3 Morpholino antisense oligonucleotides (MO1, MO2) or control Morpholino (CoMO) and in combination with the following mRNA: 2 ng preprolactin (ppl) (A–C, E), 2 ng chicken dkk3 (D), or 2 ng mouse dkk3 (F). (G–I) Whole-mount in situ hybridization of Xnot and myoD in Morpholino- injected embryos as indicated. (K–L) Statistical analysis of phenotypes is described in (B–F).
Fig. 3 Dkk3 is required for mesoderm induction. (A–C) Real-time RT-PCR analysis for the indicated marker genes of embryos injected at one- to two-cell stage with the indicated antisense Morpholinos. Embryos were analyzed at late blastula (A), early gastrula (B), or mid-gastrula stage (C). (D–F) Four-cell stage embryos were
injected into one blastomere with 3.75 ng CoMO or MO1 and either 2 ng preprolactin (ppl) or chicken dkk3 mRNAs as indicated. LacZ mRNA was co-injected as a lineage tracer and embryos were stained for b-galactosidase activity (light blue). Expression of Xbra was analyzed by in situ hybridization at stage 10.5. Note the complete gap of Xbra expression in MO1 injected cells (light blue) (E)
and the partial rescue by chicken dkk3 mRNA (F). (G) Quantitation of embryos with gaps in Xbra staining as described in (D–F).
Fig. 4 Dkk3 affects FGF and Activin/Nodal pathways. (A) Twocell stage embryos were injected into the animal pole with 50 ng of CoMO or dkk3 MO1 and animal caps were explanted at stage 9, cultured in the presence of Activin or FGF protein until sibling embryos reached stage 10.5. Induction of the indicated markers was
analyzed by real-time RT-PCR. (B) Reporter construct for FGFERK (Gal-ELK:Gal-Luc) signaling was injected, together with either 2 ng ppl, 2 ng dkk3, or 1 ng XFD mRNAs into four-cell-stage embryos as indicated. Embryos were harvested at stage 10.5 and luciferase reporter activity was measured.
Fig. 5 Smad2 phosphorylation and nuclear translocation are unaffected by Dkk3 depletion. (A) One- to two-cell-stage embryos were injected with 25 ng CoMO or dkk3 MO1, and the levels of phospho-Smad2 (pSmad2) and total Smad2/3 were analyzed by Western blot. (B) One- to two-cell-stage embryos were injected with 50 ng CoMO or dkk3 MO1, and animal caps were explanted at stage 9 and treated with Activin protein for 30 min. Animal caps were stained for nuclei with Hoechst (a–c) and analyzed by immunofluorescence for endogenous Smad2/3 (a0–c0). (C) Quantitation of Smad2/3 staining in Hoechst-positive nuclei.
Fig. 6 Dkk3 is required to maintain Smad4 levels. (A) One-cell stage embryos
were injected with antisense Morpholino oligonucleotides as indicated. At two-cell stage, embryos were re-injected with 0.15 ng/embryo Smad4. Embryos were
harvested at stage 10.5 for Western blot analysis of Smad4. (B) Two-cell stage embryos were injected with dkk3 MO1 and expression of Xectodermin was
analyzed at stage 10.5 by real-time RT-PCR. (C–F) Two- to four-cell stage embryos were injected equatorially with the following amounts of indicated mRNAs: 3 ng mdkk3 or ppl, 200 pg Xectodermin. Spina bifida or truncated
anterior structures occurred in 87% (n 5 23) and 9% (n 5 23) of embryos injected with ectodermin or ectodermin1mdkk3, respectively.
