XB-ART-16264Cell July 11, 1997; 90 (1): 181-92.
The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation.
Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians.
PubMed ID: 9230313
Article link: Cell
Genes referenced: ctrl dvl2 nog sia1
Article Images: [+] show captions
|Figure 4. Dorsal Injection of Axin mRNA Ventralizes Xenopus Embryos (a) Ventralization of Xenopus embryos by dorsal injection of Axin, and rescue by β-catenin or Siamois but not Xwnt8. Two nanograms of Axin mRNA, either alone or together with the other mRNA indicated, was injected into each of two dorsal blastomeres at the 4-cell stage. Embryos were evaluated at the tadpole stage ( Table 1), and examples are shown. The amount of Xwnt8 (20 pg), β-catenin (300 pg) or Siamois (100 pg) mRNA used was the minimal amount required to obtain full axis induction when each was injected alone in one ventral blastomere (see Figure 5a and Figure 5b). Scale bar, 1 mm. (b) Dorsal injection of Axin reduces expression of dorsal markers Siamois, Goosecoid, Chordin, and Xnr3, but not the ubiquitously expressed elongation factor EF1. Each column shows the RT-PCR analysis of a pool of uninjected embryos or embryos injected at the 4-cell stage with Axin or control β-gal mRNA (2 ng), and grown to early gastrulae. −RT, control experiments in which RNA from uninjected embryos was processed without reverse transcriptase. (c) Dorsal coinjection of β-catenin with Axin restores expression of Siamois and Goosecoid, and coinjection of Siamois restores Goosecoid expression, while coinjection of Xwnt8 has no effect. Note that the injected Siamois (not detected with the primers used in this assay) does not induce expression of endogenous Siamois.|
|Figure 5. Ability of Axin to Block Ectopic Axis Formation (a) Ventral coinjection of Axin mRNA inhibits ectopic axis formation by upstream components of the Wnt pathway (Xwnt8, Xdsh, and dnGSK-3), but not by β-catenin or Siamois, nor by Activin, Noggin or ΔBMPR. mRNA encoding the indicated dorsalizing factor was injected subequatorially in one ventral blastomere at the 4–8 cell stage, with or without 1 ng Axin, and embryos were examined for axial duplications at the late neurula–tailbud stage. The fraction of embryos with duplicated axes is indicated above each bar. mRNAs were injected in the minimal amounts needed to induce ectopic axes at high frequency: 10–20 pg Xwnt8, 1.5 ng Xdsh, 2 ng dnGSK-3, 300 pg β-catenin, 100 pg Siamois, 7.5 pg Activin, 200 pg noggin, or 1 ng ΔBMPR ( Fagotto et al. 1997). Activin-induced secondary axes were generally very incomplete. Higher amounts of Activin mRNA lead to uninterpretable phenotypes. (b) Examples of injected embryos. Scale bar, 2 mm. (c) Coinjection of Axin mRNA inhibits induction of the dorsal marker Goosecoid by Xwnt8, but not by Activin, Noggin, or ΔBMPR. Ectopic expression of Goosecoid in the ventral half of early gastrulae (stage 10 1/2) was analyzed by RT-PCR. Dorsal (D) and ventral (V) halves of uninjected embryos served as positive and negative controls (ctrl) for normal expression of Goosecoid.|
|Figure 6. Axis Duplications in Xenopus Embryos Injected Ventrally with ΔRGS and in Mouse Embryos Homozygous for the Loss-of-Function AxinTg1 Allele (a and b) Xenopus embryos with axis duplications caused by injection of 2 ng ΔRGS in one ventral blastomere (a) or 1 ng ΔRGS in two ventral blastomeres (b). The embryo in (b) is also strongly dorsalized. Scale bars, 0.5 mm. (c) Frequency of axis duplications in embryos injected with Axin, ΔRGS, or ΔRGS together with Axin or C-cadherin. (d) Ectopic expression of dorsal markers in embryos injected ventrally with ΔRGS. Each column shows the RT-PCR analysis of the dorsal (D) or ventral (V) halves of a pool of embryos. In uninjected embryos, Siamois, Goosecoid, Chordin, and Xnr3 are expressed dorsally. Ventral injection of ΔRGS, but not Axin, induces ectopic expression of the four dorsal markers. (e–g) Lateral view of a normal E7.5 mouse embryo (e) and two E8.5 AxinTg1/Tg1 embryos with axial duplications (f and g), visualized by in situ hybridization to HNF-3β, a marker of anterior axial mesoderm ( Sasaki and Hogan 1994). White arrows, primary axes; black arrowheads, ectopic axes. Scale bars, 0.2 mm.|
|Figure 7. Model for the Inhibitory Effect of Axin on Wnt Signal Transduction Established components of the Wnt pathway in the Nieuwkoop Center are indicated by blue symbols and solid black arrows, and positions where Axin might inhibit the pathway are indicated by red symbols and dashed arrows. GSK-3 promotes the degradation of β-catenin, while Wnt signals inhibit GSK-3 (via Dsh) and lead to accumulation of cytosolic β-catenin and expression of Siamois. Axin blocks the stimulation of this pathway by Wnt, Dsh, or dominant-negative GSK-3 but not by overexpression of β-catenin or Siamois. Three alternative hypotheses are illustrated: (1) Axin might inhibit a protein phosphatase (PP2A) that may otherwise dephosphorylate substrates of GSK-3; (2) Axin might stimulate the activity of GSK-3 through an unknown mechanism; (3) Axin might inhibit, via its RGS domain, the transmission of a second signal (signal 2) involving a G-protein-coupled receptor, which would otherwise stimulate the Wnt pathway downstream of GSK-3. See text for further details.|