XB-ART-5415Development 2003 Jun 01;13011:2429-41. doi: 10.1242/dev.00449.
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Xenopus Cyr61 regulates gastrulation movements and modulates Wnt signalling.
Cyr61 is a secreted, heparin-binding, extracellular matrix-associated protein whose activities include the promotion of adhesion and chemotaxis, and the stimulation of fibroblast and endothelial cell growth. Many, if not all, of these activities of Cyr61 are mediated through interactions with integrins. We explore the role of Cyr61 in the early development of Xenopus laevis. Gain- and loss-of-function experiments show that Xcyr61 is required for normal gastrulation movements. This role is mediated in part through the adhesive properties of Xcyr61 and its related ability to modulate assembly of the extracellular matrix. In addition, Xcyr61 can, in a context-dependent manner, stimulate or inhibit signalling through the Wnt pathway. These properties of Xcyr61 provide a mechanism for integrating cell signalling, cell adhesion and cell migration during gastrulation.
PubMed ID: 12702657
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: ag1 ccn1 chrd.1 fn1 gsc ins tbxt
Morpholinos: ccn1 MO1 ccn1 MO2 ccn1 MO3
Article Images: [+] show captions
|Fig. 1. Sequence and expression pattern of Xenopus Cyr61. (A) Domain structure of Cyr61. IGFBP, insulin growth factor binding protein domain; VWC, von Willebrand type C domain (also referred to as the cysteine rich domain of Chordin and short gastrulation); TSP, thrombospondin domain; CT, carboxy-terminal domain with homology to the neuronal pathfinding protein Slit. (B) Alignment of Cyr61 proteins from chick, Xenopus, rat and human. Note the high degree of conservation throughout the protein, except in the signal peptide and the variable central region. (C) Temporal expression pattern of Xcyr61 mRNA assessed by RNAase protection assay. Transcripts are present maternally and persist at least until early blastula stage 6, when they are present in both the animal (lane 6) and vegetal (lane 7) hemispheres of the embryo. Expression is then activated zygotically from mid-neurula stage 14 (lane 9). Ornithine decarboxylase (ODC) is used as a loading control. (D-F) Whole-mount in situ hybridisation analysis of Xcyr61 expression. At stage 28 (D), expression is detectable in the somites and branchial arches. A cleared embryo (E) reveals expression in the notochord, an observation that was confirmed in sectioned embryos (data not shown). At stage 34 (F), transcripts are present in the posterior cardinal vein (arrow). Sections of embryos such as these show that expression of Xcyr61 in the somites is concentrated in and around the nuclei, which suggests that transcripts are unstable (not shown). (G-I) Immunofluorescence analysis of the distribution of exogenous mouse Cyr61 in Xenopus gastrulae. (G) An uninjected embryo at early gastrula stage 10 does not react with a mouse Cyr61 antiserum. (H) An embryo previously injected with RNA encoding mouse Cyr61 reveals accumulation of mCyr61 in the blastocoel roof at the early gastrula stage (arrows). (I) Xenopus fibronectin also accumulates in the blastocoel roof (arrow). Note that expression of Xcyr61 during gastrulation proper is very low; this suggests that our morpholino oligonucleotides (Fig. 3) are targetting translation of maternal Xcyr61 mRNA. Scale bars: D, 0.4 mm; E, 0.25 mm; F, 0.4 mm; G, 0.25 mm; H, 80 μm; I, 40μ m.|
|Fig. 4. Antisense morpholino oligonucleotides directed against Xcyr61 inhibit gastrulation movements but have little effect on mesodermal specification. (A,B) Morpholino oligonucleotide MO1 causes a severe retardation in blastopore closure (B) compared with control stage 12 embryos (A). (C,D) Morpholino oligonucleotide MO1 causes a decrease in Xbra expression and shifts the Xbra expression domain towards the animal pole. Embryos are at stage 11.5. (E,F) Morpholino oligonucleotide MO1 causes expansion of the goosecoid expression domain. Embryos are at stage 11.5. (G,H) Morpholino oligonucleotide MO1 (30 ng) causes shortening of the anteroposterior axis. Embryos are at stage 35. (I-K) Bisection of embryos injected with morpholino oligonucleotide MO1 reveals changes in the structure of the blastocoel roof and of the marginal zone. (I) Embryo previously injected with a control morpholino oligonucleotide at stage 11. The blastocoel roof and marginal zone are thin and compact, as indicated by the two white lines. (J,K) Morpholino oligonucleotide MO1 (30 ng) causes a thickening of the blastocoel roof and marginal zone (lines), and a separation of cell layers (arrows). (L,M) Scanning electron microscope images of a control embryo at stage 11 (L) and an embryo at the same stage previously injected with 30 ng antisense morpholino oligonucleotide MO1 (M). Note the tightly packed epithelial appearance of the cells in the blastocoel roof of the control embryo (L), and the more loosely packed appearance of cells in the MO1-injected embryo, with some cells apparently about to detach (arrows; M). Note also that the migration of the large flat mesendodermal cells visible at the bottom of (L) is impaired in MO1-injected embryos (M). (N,O) Morpholino oligonucleotide MO2 causes a decrease in fibronectin assembly in the blastocoel roof. (N) Fibronectin forms an elaborate fibrillar network in the blastocoel roof of control embryos. (O) Fibronectin assembly is reduced in the blastocoel roof of morpholino-injected embryos. (P) Western blot analysis indicates that levels of fibronectin are similar in control and morpholino-injected embryos. HSP-70 was used as a loading control. Scale bars: in L, 100 μm for L,M; in O, 100 μm for O,N.|
|Fig. 10. Xcyr61 induces formation of cement glands in Xenopus animal caps and synergises with a truncated BMP receptor (tBR) to induce additional heads. (A) Xenopus embryo at stage 32 showing expression of the cement gland marker XAG1. (B) Induction of XAG1 in animal caps by Xcyr61. (C) Induction of a partial secondary axis by tBR (15 out of 46 cases). (D) Induction of a partial secondary axis by Xcyr61 (19 out of 90 cases). (E) Induction of an additional head by co-expression of tBR and Xcyr61 (18 out of 83 cases; an additional 18 embryos displayed partial secondary axes). Muscle in C and D is marked by monoclonal antibody 12/101.|