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First published online April 22, 2004
doi: 10.1242/10.1242/dev.01045

Development 131, 2137-2147 (2004)
Published by The Company of Biologists 2004
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Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex

Sara Mercurio1,*, Branko Latinkic1,{dagger}, Nobue Itasaki2, Robb Krumlauf2,{ddagger} and J. C. Smith1,*,§

1 Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK



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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, 1975Go). 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.

 


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  		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.

 


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  		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 CTGF{Delta}CT 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.

 


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  		Fig. 4. Inhibition of WNT signalling requires the CT domain of CTGF. (A) Domain structures of CTGF and CTGF{Delta}CT. (B-D) CTGF{Delta}CT cannot inhibit induction of secondary axes by Xwnt8. (B) Secondary axis induced by Xwnt8. (C) Inhibition of secondary axis formation by CTGF. (D) CTGF{Delta}CT cannot inhibit secondary axis formation. (E) CTGF{Delta}CT 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 CTGF{Delta}CT 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) CTGF{Delta}CT 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 Ctgf{Delta}CT RNA.

 


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  		Fig. 5. CTGF interacts with the WNT receptor complex. In all experiments, proteins were provided in the form of conditioned medium, prepared as described in Materials and methods. Media were mixed as indicated, precipitated by Protein A Sepharose beads, and visualised by immunoblotting with the indicated antibodies. (A) CTGF interacts with LRP6 and Frizzled8 extracellular domains (LRP6N and FzCRD) under low stringency conditions (150 mM NaCl). Binding to LRP6, but not to Frizzled8, resists more stringent washing (500 mM NaCl) (top panel). Input levels of CTGF (middle panel) and the IgG-tagged proteins (bottom panel) are shown. (B) The CT domain is required for the interaction of CTGF with LRP6. CTGF but not CTGF{Delta}CT is precipitated by LRP6N (top panel). Levels of CTGF and CTGF{Delta}CT (middle panel) and of control IgG, Fz8CRD and LRP6N (bottom panel) prior to immunoprecipitation are shown. (C) CTGF binds to both EGF repeats 1, 2 and 3, 4 of LRP6. LRP6 extracellular domain (LRP6N) and two deletion constructs, LRP6N{Delta}1,2 and LRP6N{Delta}3,4, all interacted with CTGF whereas control IgG did not (top panel). Note that whereas CTGF protein levels are comparable in all lanes prior to immunoprecipitation (middle panel), LRP6N{Delta}3,4 protein levels are lower than both LRP6N and LRP6N{Delta}1,2 (bottom panel), indicating that the interaction of CTGF with this deletion is stronger than appears in the top panel. (D) DKK1 interacts with EGF repeats 3 and 4 of LRP6. DKK1 binds to LRP6N and LRP6N{Delta}1,2, but not to LRP6N{Delta}3,4 (top panel). Dkk-(middle panel) and IgG-tagged protein levels (bottom panel) prior to immunoprecipitation are shown.

 

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© The Company of Biologists Ltd 2004