Mercurio S et al. (2004), Connective-tissue growth factor modulates WNT s...

XB-ART-3647

Development 2004 May 01;1319:2137-47. doi: 10.1242/dev.01045.

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Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex.

Mercurio S , Latinkic B , Itasaki N , Krumlauf R , Smith JC .

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Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix, they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be because of its recently identified ability to interact with BMP4 and TGF beta. We demonstrate, using Xenopus embryos, that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF, which is distinct from the BMP- and TGF beta-interacting domain. Our results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis.

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Species referenced: Xenopus laevis
Genes referenced: actl6a bmp4 ccn1 ccn2 dvl2 ins lrp6 mlc1 nodal3.2 odc1 otx2 pax6 sia1 slit1 snai2 sox3 tgfb1 tubb2b wnt8a
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	Fig. 1. CTGF and its expression pattern. (A) Schematic representation of the domain structure of CTGF. Shown below is an alignment of the CT domains of xCTGF, XCyr61, XSlit and XWiseA. The eight conserved cysteines are indicated by a dot and amino acids conserved in at least three out of the four proteins are shaded grey. The CT domain of xCTGF shows 52% amino acid identity with XCyr61, 27% with Xslit, and 19% with XWiseA. (B) Temporal expression pattern of Ctgf studied by reverse transcription-polymerase chain reaction. Stages are according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1975). Ornithine decarboxylase (ODC) acts as a loading control. Note the absence of maternal expression of CTGF. (C-H) In situ hybridisation analysis of CTGF expression. (C) Stage 20. Expression is detectable in the somites and axial midline. (D) Stage 25. Expression persists in somites and axial midline. (E) Stage 28. Expression is detectable in brain and branchial arches. (F) Stage 32. CTGF is expressed in the heart, in distinct domains in the brain, in the floorplate and in the hypochord. (G,H) Sections through a stage 32 embryo confirm expression of Ctgf in floorplate, heart and somites. CT, C-terminal cystine knot; IGFB, Insulin growth factor binding domain; Signal, signal peptide; TSP-1, thromospondin type 1 repeat; CR, Von Willebrand type C repeat.
	Fig. 2. The effects of overexpression of CTGF resemble those caused by inhibition of WNT signalling. (A) Overexpression of CTGF (bottom embryo) causes a shortening of Xenopus embryos. Three independent experiments were performed with similar results each time. (B,C) Compared with controls (B), CTGF-injected embryos have an enlarged cement gland (C). (D-K) Unilateral overexpression of CTGF causes changes in gene expression that resemble those caused by inhibition of WNT signalling. (D) The expression domain of Otx2 is expanded. (E) CTGF causes a down-regulation of the future hindbrain domain of Pax6 and slightly expands the anterior domain. (F) CTGF causes down-regulation of N-tubulin in the neural plate. (G) The expression domain of Xsox3 in the neural plate is expanded. (H,I) Compared with the control side of the embryo (H), CTGF causes down-regulation of XSox3 in the dorsolateral placode (I). (J) CTGF causes down-regulation of Slug. (K) The muscle-specific gene myosin light chain 1 (MLC1) is down-regulated by Ctgf. I: injected; NI: Not injected. The injected sides of embryos are identified by pale blue lacZ staining.
	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 CTGFδCT 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.
	Fig. 4. Inhibition of WNT signalling requires the CT domain of CTGF. (A) Domain structures of CTGF and CTGFδCT. (B-D) CTGFδCT cannot inhibit induction of secondary axes by Xwnt8. (B) Secondary axis induced by Xwnt8. (C) Inhibition of secondary axis formation by CTGF. (D) CTGFδCT cannot inhibit secondary axis formation. (E) CTGFδCT is a poor inhibitor of Xwnt8-induced activation of the TOPFLASH reporter; CTGF cannot inhibit activation of the TOPFLASH reporter by Dishevelled. Both blastomeres of Xenopus embryos at the 2-cell stage received injections of 20 pg TOPFLASH DNA, 10 pg pRLTK DNA and the indicated combinations of 1 ng CTGF RNA, 1 ng CTGFδCT RNA, 50 pg Xwnt8 RNA and 1 ng Dishevelled RNA. Animal caps were dissected at stage 8, and 20 caps per sample were assayed for Firefly and Renilla 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. (F-H) CTGFδCT cannot inhibit activin-induced elongation of Xenopus animal caps. (F) Animal caps treated with 8 units/ml of activin protein undergo elongation. (G) Animal caps derived from embryos injected with 1 ng Ctgf RNA do not undergo elongation. (H) Elongation of animal caps is not inhibited by 1 ng of CtgfδCT RNA.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorso-lateral view, anterior left.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 28,lateral view, anterior left, dorsla up.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32 ,lateral view, anterior left, dorsal up.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32 , transverse section through heart region, dorsal left.
	ccn2 (cellular communication network factor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, horizontal section through trunk region, dorsal left.