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BACKGROUND: Recent studies have demonstrated that the Wnt, Frizzled and Notch proteins are involved in a variety of developmental processes in fly, worm, frog and mouse embryos. The Dishevelled (Dsh) protein is required for Drosophila cells to respond to Wingless, Notch and Frizzled signals, but the molecular mechanisms of its action are not well understood. Using the ability of a mutant form of the Xenopus homologue of Dsh (Xdsh) to block Wnt and Dsh signalling in a model system, this work attempts to clarify the role of the endogenous Xdsh during the early stages of vertebrate development.
RESULTS: A mutant Xdsh (Xdd1) with an internal deletion of the conserved PDZ/DHR domain was constructed. Overexpression of Xdd1 mRNA in ventral blastomeres of Xenopus embryos strongly inhibited induction of secondary axes by the wild-type Xdsh and Xwnt8 mRNAs, but did not affect the axis-inducing ability of beta-catenin mRNA. These observations suggest that Xdd1 acts as a dominant-negative mutant. Dorsal expression of Xdd1 caused severe posterior truncations in the injected embryos, whereas wild-type Xdsh suppressed this phenotype. Xdd1 blocked convergent extension movements in ectodermal explants stimulated with mesoderm-inducing factors and in dorsal marginal zone explants, but did not affect mesoderm induction and differentiation.
CONCLUSIONS: A vertebrate homologue of Dsh is a necessary component of Wnt signal transduction and functions upstream of beta-catenin. These findings also establish a requirement for the PDZ domain in signal transduction by Xdsh, and suggest that endogenous Xdsh controls morphogenetic movements in the embryo.
Figure 4. Early marginal zone markers and mesodermal tissue differentiation are not affected by Xdd1. (a) Whole-mount immunochemical staining with muscle-specific 12/101 antibodies. Top: control embryo; bottom: two embryos injected dorsally with Xdd1 mRNA. Arrowhead points to a somite. (b) Cross-section of an Xdd1-injected embryo after whole-mount staining with notochord-specific MZ15 antibodies. Brown staining outlines a sheath around notochord (nc). Bar in (b), which also refers to (d), is 150 μm. (c) Whole-mount staining with MZ15 antibodies. Top left: four embryos injected with Xdd1 mRNA; white arrowhead points to notochord. Bottom left: two embryos injected with wild-type Xdsh mRNA; control uninjected embryo is shown on the right. (d) Cross-section of a control uninjected embryo stained with MZ15 antibodies. (e) Northern analysis of early marginal zone markers in control embryos (lane 1) and in embryos injected dorsally with Xdd1 (lane 2) or Xdsh (lane 3) mRNAs. Total RNA was prepared when control embryos reached stage 10.5. Xbra is a general marginal zone marker. Xotx2, goosecoid (Gsc), Xlim 1 and chordin are dorsal marginal zone markers. Xnr3 is expressed in the superficial layer of the Spemann organizer. Fibronectin (FN) mRNA is a control for loading. (f) Xdd1 does not alter the localization of chordin transcripts. In situ hybridization of stage 10.5 gastrula embryos with chordin anti-sense RNA probe. Far left: uninjected albino embryo; centre left: uninjected pigmented embryo; right: two pigmented embryos injected with Xdd1 mRNA in the dorsal margin at the two- to four-cell stage.
Figure 1.
The effect of Xdd1 mRNA on axis-inducing ability of Xwnt8 and Xβcat mRNAs. One ventro-vegetal blastomere of 4–8 cell embryos was injected with (a) 2 pg of Xwnt8 mRNA, (b) 2 pg of Xwnt8 mRNA plus 1.5 ng of Xdd1 mRNA, (c) 0.8 ng of Xβcat mRNA of (d) 0.8 ng of Xβcat mRNA plus 1.5 ng of Xdd1 mRNA.
Figure 2.
Embryos microinjected with Xdd1 mRNA develop a specific morphogenetic defect. Two-cell embryos were injected dorsally with 1 ng of each mRNA into each blastomere. (a) Stage 14 neurulae injected with Xdd1 mRNA (on the right) and with Xdd2 mRNA (on the left). Note the failure of neural folds (shown by two small white arrowheads) to close into a neural tube. The cement gland develops at the anterior end (large arrowhead); the closed blastopore is visible at the posterior end. The embryo injected with Xdd2 mRNA has a normal neural tube and is indistinguishable from uninjected embryos and from embryos injected with wild-type Xdsh (not shown). (b) Stage 40 tadpoles. Top: control embryo (uninjected); bottom: Xdd1-injected embryos with posterior deficiencies. (c) Sagittal histological section of Xdd1-injected embryo (stage 40); (d) sagittal section of a control embryo (stage 40). Abbreviations: m, muscle; nc, notochord; nt, neural tissue; cg, cement gland. The bar in (d), which also refers to (c), is 200μm.
Figure 3.
Wild-type Xdsh mRNA corrects dorso-posterior deficiencies caused by injections of Xdd1 mRNA. Injections of wild-type Xdsh (top three embryos), Xdd1 (middle row) and a mixture of both Xdd1 and wild-type Xdsh (bottom row) were performed as described in Figure 2. A control uninjected embryo is shown on the left.
Figure 5.
Xdd1 inhibits morphogenetic movements of ectodermal explants stimulated with activin. (a) Morphology of explants at stage 17. Top left: control uninduced explants; top right: control explants induced with activin; bottom left: explants overexpressing Xdd1; bottom right: explants overexpressing Xdd1 and stimulated with activin. No difference is apparent between control uninjected explants and explants injected with Xdd2 (data not shown). (b) Northern analysis of early markers at the early gastrula stage (stage 10.5). Lanes 1,2: uninjected explants; lanes 3,4: Xdd1-injected explants; lane 5, control embryos. Treatment of explants with activin was as indicated. Total RNA was prepared when control embryos reached stage 10.5 Xbra is a general marginal zone marker. Xlim1 marks dorsal, and Xwnt8 marks ventrolateral marginal zone. Fibronectin (FN) mRNA is a loading control.
Figure 6.
The effect of Xdd1 on cell movements may depend on cell–cell interactions. (a)Xdd1 inhibits morphogenetic movements of the dorsal marginal zone. Dorsal marginal zone explants were isolated at stage 10 from embryos that had been injected earlier with 1 ng of Xdd1 mRNA (on the left) or Xdd2 mRNA (on the right). (b,c) Lineage tracing of Xdd1-microinjected embryos. (b) Embryos were injected at eight-cell stage into the dorso-animal blastomere with β-gal mRNA only (bottom embryo) or with β-gal mRNA plus Xdd1 mRNA (two top embryos). (c) Two top dorso-animal blastomeres at the 32-cell stage were injected with 5 nl of β-gal RNA (two top embryos) or Xdd1 plus β-gal RNAs (three bottom embryos). The arrowhead points to the area in which developmental abnormalities are observed, despite the absence of Xdd1 and β-gal RNA (unstained). Embryos were cultured until stage 35, then fixed and β-gal-expressing cells were visualized histochemically.
Figure 7.
(a) Interaction of Dishevelled with other components of the Wnt signal transduction pathway. Dishevelled is thought to modulate the activity of β-catenin/Armadillo by transducing a signal from the Frizzled/Notch receptors to the GSK3/APC/β-catenin complex. This modulation leads to subsequent transcriptional activation of the target genes. (b) The wild-type and mutated Xdsh constructs used in this study.
Figure 7. (b) The wild-type and mutated Xdsh constructs used in this study.
Figure 7. (b) The wild-type and mutated Xdsh constructs used in this study.
