Rothbächer U et al. (1995), Functional conservation of the Wnt signaling pa...

XB-ART-19419

Dev Biol 1995 Aug 01;1702:717-21. doi: 10.1006/dbio.1995.1249.

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

Rothbächer U , Laurent MN , Blitz IL , Watabe T , Marsh JL , Cho KW .

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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.

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Species referenced: Xenopus
Genes referenced: actc1 dvl1 dvl2 fgf2 gsc

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FIG. 1. Ectopic expression of Drosophila dsh induces secondary axes by changing the fate of ventral marginal zones (VMZs) to that of dorsal marginal zones (DMZs). (A) Embryo injected with Drosophila dsh mRNA into ventral blastomeres of the 4-cell stage exhibits induction of a secondary axis. Anterior is left and dorsal is up. The secondary axis with dark pigment is indicated with an arrow. Note, however, the absence of anterior-most structures in the secondary axis. The partial phenocopying effect of Drosophila d:sh may be attributed to the heterospecific nature of the dsh molecule used in this experiment or to the possibility that dsh-mediated signaling constitutes only part of the Wnt signaling cascade in Xenopus dorsal-ventral patterning. (B) Transverse section through trunk of a dsh-injccted embryo. Note the enlarged somite (SM) to the right (closer to the secondary neural tissue) compared to the somite of the left Dashed lines mark the somite boundary. Abbreviations: N, notochord; 1 °NT, primary neural tissue; 2°NT, secondary neural tissue; SM, somite. (C) Schematic diagram showing the injection assay and isolation of explants. Drosophila d.sh rnRNA was injected in the equatorial region of the two ventral b].astomeres at the 4-ce]J stage. VMZs and DMZs were dissected at early gastrula of stage 10.25 and cultured until uninjected sibling embryos reached the indicated stages and RNA was isolated for analysis by RT-PCR assays. Some injected embryos were also grown to stage 30 for phenotypic examination. (D) RT-PCR of RNA from explants injected with d.c;;h. DMZ (D) ~nd VMZ (V) explants from embryos injected ventrally with d.~h (+)were compared to uninjected controls (-) at stage 11 (gastrula, lanes 1-4) and stage 25 (tadpole, lanes 5-8) equivalents. PCR was performed to detect the expression of goosecoid, orthodenticle 2, cardiac actin, and N-CAM. Histone H4 primers were included as controls showing that the amount of amplification does not vary significantly between samples. The entire experiment was performed twice with the same results.

FIG. 2. Specificity of Drosophila dsk in Xenopus Wnt signaling. (A-F) Drosophila rl~h. synergizes with bFGF. ·while uninjected control animal cap tissues remain ectoderm (A), they differentiate into ventral type mesoderm and do not elongate after bFGF treatment (100 ~g/ml) (B). Animal caps receiving either Xwnt8 or dsh mRNA alone (200 pg/embryo) failed to elongate (C and E), whereas animal caps that received dsh or Wnt mRNA together with bFG F treatment became elongated (D and F). The synergism of dRh with bFG F was observed using dsh concentrations as low as 40 pg per embryo. (G-1) dsh activates a Wnt-responsive reporter gene. (G) Schematic map showing the gsc WT/ Luc and M4/ Luc constructs. Note that the activin-responsive element (ARE) and Wnt-responsive element (WRE) are physically distinct and separable. M4/ Luc contains a 6-bp substitution in the ARE. (H) The mutations in M4/Luc inactivated the ARE. After rnicroinjecting either the gsc WT/Luc or M4/ Luc construct into 4·cell stage embryos (20 f.Lg/ml), animal caps were isolated at blastula stage and incubated with or without a.ctivin. After 3 hr, animal caps were homogenized in 50 mMTris (pH 7.5) and reporter gene activities (luciferase) were measured to quantitate the relative induction of the gsc promoter by activin. While the WT/ Luc was induced over 20-fold by activin treatment, the M4/Luc was not induced. (I) The M4/Luc gene is induced by both Xwnt8 and dsh. M4/Luc was either injected alone or co-injected with the indicated mRNAs into either the dorsal or the ventral side of 4·cell stage embryos. Dorsal and ventral marginal zones were removed at early gastrula (stage 10.25), incubated for 1 hr, homogenized as above, and assayed for Juciferase activity. Comparison of dorsal and ventral ex plants injected with M4 alone revealed that the dorsal explants induce the M4 reporter gene 5- to 10-fold higher than the ventral explants. Co-injection of Xwnt8 mRNA caused a 10-fold induction of M4 in ventral tissues, which is indicative of dorsalization of these tissue. Activation of M4/ Luc was also seen in ventral explants injected with dsh. Activation of M4/Luc in the dorsal explants were similar whether the tissue received Xwnt-8, dsh, or M4 alone. The experiments using ventral explants were repeated four times and essentially identical relative activation by Xwnt8 and dsh was observed in all cases.