Fig. 3. CTGF inhibits WNT signalling. (A-D) Induction of duplicated axes by Xwnt8 is inhibited by CTGF. (A) Control embryos at stage 28. (B-D) Embryos were injected at the 4-8-cell stage into one ventral-vegetal blastomere with RNA encoding CTGF alone (B), Xwnt8 alone (C), or both (D). Secondary axis induction is inhibited by CTGF. (E) CTGF inhibits induction of Xnr3 and Siamois by Xwnt8. Ventral marginal zone (VMZ) or dorsal marginal zone (DMZ) tissue was isolated from Xenopus embryos at early gastrula stage 10. Some embryos had previously been injected with RNA encoding Xwnt8 or CTGF or both, as indicated. Expression of Xnr3, Siamois or, as a loading control, ornithine decarboxylase (ODC) was assayed by RT-PCR. –RT: No reverse transcription control. (F) CTGF inhibits induction of the TOPFLASH reporter by Xwnt8. Xenopus embryos at the 2-cell-stage were injected into both blastomeres with 20 pg TOPFLASH (firefly luciferase) DNA, 10 pg pRLTK (Renilla luciferase) DNA and, where indicated, 1 ng CTGF RNA or 50 pg Xwnt8 RNA or a combination of the two. Animal caps were dissected at stage 8, and 20 caps per sample were assayed for Luciferase activities 3 hours later. Firefly luciferase activities were then normalised to Renilla activities. This experiment represents a typical result out of three independent experiments. (G-J) CTGF inhibits activin-induced elongation of animal caps. (G) Control embryo at stage 18. (H) Control animal caps at the equivalent of stage 18 remain spherical. (I) Activin-treated animal caps elongate. (J) Elongation is inhibited by overexpression of CTGF. (K-N) CTGF inhibits the elongation of isolated dorsal marginal zone regions. Dorsal marginal zone regions were isolated from control embryos (K) or from embryos injected with 1 ng (M) or 2.5 ng (N) Ctgf RNA. Ventral marginal zone regions (L) acted as controls. Explants were cultured to the equivalent of stage 15 and scored as described in Table 1. Note that CTGF causes a reduction in the elongation of dorsal marginal zone regions. (O) CTGF and CTGFCT do not inhibit activin-induced expression of muscle-specific actin in Xenopus animal caps. Expression of muscle-specific actin and ODC was assessed by RT-PCR. –RT: No reverse transcription control.