Axis determination by inhibition of Wnt signaling in Xenopus

Abstract

The Wnt family of secreted polypeptides participate in a variety of developmental processes in which embryonic polarity is established. To study a role for Wnt ligands in vertebrate axis determination, we interfered with Wnt signaling in the embryo using the extracellular domain of Xenopus Frizzled 8 (ECD8), which blocks Wnt-dependent activation of a target gene in Xenopus ectodermal explants. Expression of ECD8 in ventral blastomeres resulted in formation of secondary axes containing abundant notochord and head structures. These results suggest that Wnt signaling is required to maintain ventral cell fates and has to be suppressed for dorsal development to occur.

Figure 1

ECD8 is a potent Wnt inhibitor. Two-cell embryos were injected into animal pole with ECD8 (2 ng), frzb (2 ng), Xwnt3a (5 pg), Xwnt8 (2.4 pg), Xwnt2b (5 pg), Xwnt5a (0.6 ng), and Xfz8 (0.6 ng) mRNA, or in combination as indicated. Animal cap explants were prepared from the injected embryos when sibling control reached stage 8 and cultured until control embryos reached stage 10.5, at which time total RNA was isolated for Northern analysis. Each gel lane contains RNA isolated from ten explants or two embryos. Xnr3 and gsc are organizer markers. fibronectin (FN) serves as a control.

Figure 2

Developmental effects of ECD8. ECD8 mRNA (1–2 ng) was injected into one ventrovegetal blastomere (A–C) or into two dorsovegetal blastomeres (D,E) at the four- to eight-cell stage. External morphology of injected embryos (A,D). (Arrowheads) Induced secondary axes; (arrows) enlarged or fused cement glands. (D) An uninjected sibling embryo (stage 33) is shown at bottom. Histological analysis of injected embryos in transverse (B,C) and sagittal (E) sections. (F) Transverse section of a control sibling embryo. (1°), primary axis; (2°) secondary axis; (nt) neural tissue; (e) eye; (cg) cement gland; (nc) notochord; (mu) muscle. Scale bar, 400 μm for E and 200 μm for B,C, and F.

Figure 3

Lineage tracing of embryos overexpressing ECD8 and frzb RNAs. Subequatorial region of a ventral blastomere of four cell embryos was injected with 1 ng of ECD8 (A,C) or frzb (B,D) mRNA together with 0.5 ng of lacZ mRNA. When control siblings reached stage 39, the injected embryos were fixed and stained for β-galactosidase activity. (A) Staining is predominantly observed in the secondary head (arrowheads). (B) Embryos injected with Frzb mRNA developed kinked tails that were stained on the injected side (left). The uninjected side of two other embryos is shown right. (C) Transverse section of an embryo injected with ECD8 RNA reveals β-gal staining in neural tissue, cement gland, and epidermis of the secondary head, but notochord and somites remain unstained. (D) Transverse section of an embryo injected with frzb RNA shows stained lateral plate mesoderm, neural tube, and epidermis on one side. Abbreviations are as in Fig. 2, except for (lp) lateral plate mesoderm. Scale bar in C (also refers to D), 200 μm.

Figure 4

Selective effect of ECD8 on marginal zone markers. Two ventrovegetal blastomeres of four- to eight-cell embryos were injected with 5 pg of Xwnt8 mRNA, 2 ng of ECD8 mRNA, or with both mRNAs as indicated. Dorsal and ventral halves were dissected at the beginning of gastrulation and collected immediately (A) or cultured until stage 12.5 (B) or stage 16 (C) for Northern analysis. chordin (Chd), cerberus (Cerb), Xlim1, Xnr3, gsc, and frzb are organizer markers; XANF1 and Xotx2 are dorsal marginal zone and anterior ectoderm markers; Xwnt8 and PV.1 are ventrolateral markers; myoD is an early marker for somites; XAG1 is a cement gland marker; NCAM and XIF3 are general neural markers. fibonectin (FN) serves as a loading control. The two bands at top revealed by the cardiac actin probe correspond to cytoskeletal actin and reflect loading (C); the band at bottom is muscle specific.

Figure 4

Selective effect of ECD8 on marginal zone markers. Two ventrovegetal blastomeres of four- to eight-cell embryos were injected with 5 pg of Xwnt8 mRNA, 2 ng of ECD8 mRNA, or with both mRNAs as indicated. Dorsal and ventral halves were dissected at the beginning of gastrulation and collected immediately (A) or cultured until stage 12.5 (B) or stage 16 (C) for Northern analysis. chordin (Chd), cerberus (Cerb), Xlim1, Xnr3, gsc, and frzb are organizer markers; XANF1 and Xotx2 are dorsal marginal zone and anterior ectoderm markers; Xwnt8 and PV.1 are ventrolateral markers; myoD is an early marker for somites; XAG1 is a cement gland marker; NCAM and XIF3 are general neural markers. fibonectin (FN) serves as a loading control. The two bands at top revealed by the cardiac actin probe correspond to cytoskeletal actin and reflect loading (C); the band at bottom is muscle specific.

Figure 5

The effect of ECD8 on BMP signaling. Animal caps were prepared as in Fig. 1 from embryos injected with 2 ng of ECD8, 0.4 ng of BMP4, or 2 ng of tBR mRNA as indicated. The animal caps were harvested when control siblings reached stage 10.5 (A), 12.5 (A), or 25 (B) for Northern analysis. fibronectin (FN) and EF1α are loading controls. Xbra is a general marginal zone marker; PV.1 is a ventrolateral marker; XIF3 and NCAM are pan-neural markers. (C) The effect of ECD8 on signaling from CABR. Each blastomere of four-cell embryos was injected with 20 pg of Xvent2–luc DNA and 0.5 ng of tBR, CABR, and ECD8 RNAs as indicated. Luciferase activity was measured in injected embryos at stage 10. Experimental data are expressed as the means from triplicate samples +/− standard deviations.

Figure 5

The effect of ECD8 on BMP signaling. Animal caps were prepared as in Fig. 1 from embryos injected with 2 ng of ECD8, 0.4 ng of BMP4, or 2 ng of tBR mRNA as indicated. The animal caps were harvested when control siblings reached stage 10.5 (A), 12.5 (A), or 25 (B) for Northern analysis. fibronectin (FN) and EF1α are loading controls. Xbra is a general marginal zone marker; PV.1 is a ventrolateral marker; XIF3 and NCAM are pan-neural markers. (C) The effect of ECD8 on signaling from CABR. Each blastomere of four-cell embryos was injected with 20 pg of Xvent2–luc DNA and 0.5 ng of tBR, CABR, and ECD8 RNAs as indicated. Luciferase activity was measured in injected embryos at stage 10. Experimental data are expressed as the means from triplicate samples +/− standard deviations.

Figure 6

The effect of ECD8 on ectodermal explants treated with mesoderm-inducing factors. Animal caps were isolated at the midblastula stage from uninjected embryos and from embryos injected with 2 ng of ECD8 mRNA. Explants were treated with 100 ng/ml bFGF or 5 ng/ml activin and cultured until stage 10.5 (A) or stage 25 (B) for Northern blot analysis. Molecular markers are described in legends to Figs. 1, 4, and 5. (Em) Sibling embryos.

Figure 6

The effect of ECD8 on ectodermal explants treated with mesoderm-inducing factors. Animal caps were isolated at the midblastula stage from uninjected embryos and from embryos injected with 2 ng of ECD8 mRNA. Explants were treated with 100 ng/ml bFGF or 5 ng/ml activin and cultured until stage 10.5 (A) or stage 25 (B) for Northern blot analysis. Molecular markers are described in legends to Figs. 1, 4, and 5. (Em) Sibling embryos.

Figure 7

Inhibition of dorsoanterior development by Xwnt3a. Two animal dorsal blastomeres at four- to eight-cell embryos were injected with 4 pg of Xwnt3a (A) or 2.5 pg of Xwnt8 mRNA (B). Morphology of injected embryos is shown. (C) Control siblings at comparable stage (stage 40).