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doi: 10.1242/10.1242/dev.00449

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Xenopus Cyr61 regulates gastrulation movements and modulates Wnt signalling

¹ Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Wellcome Trust/Cancer Research UK Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
³ Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607-7170, USA

View larger version (74K): [in a new window]   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, 40 µm.

View larger version (86K): [in a new window]   Fig. 2. Effects of overexpression of Cyr61 and similarity with the phenotype obtained following injection of antisense morpholino oligonucleotides. (A-C) Control stage 21 (A), Xcyr61-(B) and MO1-injected (C) embryos. Note the disruption of gastrulation. (D-F) Intra-blastocoelic injections of BSA (D) and mouse Cyr61 (E,F). Note the similarity of the phenotypes in E,F to those in B,C. (G-I) Analysis of isolated dorsal marginal zone regions highlights defects in epiboly caused by overexpression (500 pg RNA; H) or downregulation (I) of Xcyr61. Note that the endoderm in H and I is not covered by epidermis (arrows). Scale bar in A: 0.4 mm for A-I.

View larger version (30K): [in a new window]   Fig. 3. (A) Comparison of the sequences of the 5' untranslated regions of the two Xcyr61 pseudoalleles Xcyr61a and Xcyr61b, indicating the sequences targeted by the antisense morpholino oligonucleotides MO1 (purple), MO2 (blue) and MO3 (red). The translation start site (atg) is underlined. (B) The Xcyr61 antisense morpholino oligonucleotides (at 0.3 and 0.75 µg/µl) block in vitro translation of Xcyr61, but not that of goosecoid. Three control MO oligonucleotides (C1-3; 0.75 µg/µl) do not affect translation of either protein.

View larger version (112K): [in a new window]   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.

View larger version (30K): [in a new window]   Fig. 5. Partial rescue of the effects of antisense morpholino oligonucleotide MO2 by injection of Xcyr61 mRNA lacking the 5' untranslated region against which MO2 is directed. Injection of MO2 causes a range of defects, including gastrulation defects, truncation of the axis and small eyes. Xcyr61 mRNA can ameliorate the small eye phenotype but not the gastrulation defect, which may be more sensitive to levels and precise localisation of Xcyr61.

View larger version (97K): [in a new window]   Fig. 6. Cyr61 promotes CT domain- and heparan sulphate proteoglycan-dependent spreading of cells from blastulae; Xcyr61 is required for cell-cell adhesion. (A,B) Confocal microscope images of phalloidin-FITC stained activin-treated animal pole blastomeres spreading on fibronectin (A) or purified mouse Cyr61 (B). Cells plated on fibronectin have a polarised phenotype, with a least one long filopodium and, at the opposite end of the cell, lamellipodia. Cells plated on Cyr61 are characterised by extensive lamellipodia and no filopodia. (C-H) Phase-contrast images of live activin-treated animal pole blastomeres seeded on bovine serum albumin (BSA), fibronectin (FN), Cyr61 (CYR61) or Cyr61 lacking the CT domain (–CT) in the absence (C,D,E,G) or presence (F,H) of heparin (H). Cell spreading on Cyr61 requires the CT domain of that protein and is inhibited by heparin. Cell spreading on fibronectin is not inhibited by heparin. (I,J) Re-aggregation of blastomeres requires Xcyr61. Blastocoel roofs derived from control embryos or from embryos injected with MO2 (30 ng) were dissociated and allowed to re-aggregate. Cells derived from control embryos formed large clumps (I); those derived from MO2-injected embryos re-aggregated poorly (J). Scale bars: in B, 20 µm for A,B; in C, 100 µm for C-H; in I, 300 µm for I,J.

View larger version (34K): [in a new window]   Fig. 7. Secondary axis and Wnt/ß-catenin pathway activation by Xcyr61. (A-F) Xcyr61 mRNA (500 pg) was injected into ventral cells of Xenopus embryos at the four-cell stage. (A) Partial secondary axis induced by Xcyr61 (arrow). (B) Complete secondary axis, with secondary head and notochord, revealed by MZ15 antibody staining. (C-F) Dorsalisation caused by Xcyr61, in decreasing order of severity. Muscle is visualised by a cardiac actin probe. Arrows indicate the effects of Xcyr61. (C) Partial secondary axis, (D) secondary muscle outgrowth, (E) split somite and (F) isolated muscle cells at ectopic location. (G) Xcyr61 induces expression of Siamois, a direct target of Wnt/ß-catenin signalling, in ventral marginal zone tissue. Siamois is expressed at high levels in dorsal marginal zone tissue and is also induced in ventral marginal zone cells by Xwnt8. Ornithine decarboxylase acts as a loading control. (H-K) Xcyr61, and a deletion comprising only domains 1 and 2 (IGFBP and VWC) of the protein, causes dorsalisation of ventral marginal zone tissue. Ventral marginal zone tissue was dissected from embryos that had previously been injected with the indicated Xcyr61 constructs. They were allowed to develop to the equivalent of stage 32 when they were assayed for expression of cardiac actin. A control dorsal marginal zone explant is included for comparison. (L) Like Dishevelled (Dsh), Xcyr61 can activate of the TOPFLASH reporter, albeit weakly. Activity resides in the IGFBP domain (domain 1).

View larger version (43K): [in a new window]   Fig. 8. Xcyr61 can antagonise the Wnt pathway. (A) Constructs used in these experiments. (B-H) Antagonism of Xwnt8-induced secondary axes (arrows) was obtained with full-length Xcyr61 and a construct comprising only domain 4 (CT), but inhibition was not observed with a construct comprising domains 1 and 2 (IGFBP and VWC). In all experiments Xwnt8 mRNA (20 pg) was injected ventrally either alone or together with RNA encoding Xcyr61 (500 pg), Xcyr61 (1,2; 500 pg), or Xcyr61 (4; 2 ng). (I,J) Xcyr61 blocks induction of the TOPFLASH reporter by Xwnt8 (I) but inhibits induction by Dishevelled (I) and ß-catenin (J) only weakly. Luciferase assays were performed on animal caps injected with the indicated RNAs (600 pg) together with 10 pg each of the TOPFLASH reporter and pRL-TK as a standardisation control.

View larger version (95K): [in a new window]   Fig. 9. Overexpression of Xcyr61 blocks activin-induced convergent-extension movements but not mesoderm induction. Inhibition of Xcyr61 function does not inhibit extension. (A-D) Animal pole explants are shown at stage 17, when activin induced elongation (B) is most obvious. Injection of full-length Xcyr61 (500 pg; C), but not Xcyr61 (1,2,3; 500 pg; D), blocks elongation. Uninjected control animal caps are shown in (A). (E-J) Animal caps allowed to develop to the equivalent of stage 32 and analysed for expression of cardiac actin. (E) Control animal caps. (F) Animal caps derived from embryos injected with RNA encoding Xcyr61. (G) Animal caps derived from embryos injected with RNA encoding Xcyr61 (1,2,3). (H) Activin-treated animal caps. (I) Activin-treated animal caps derived from embryos injected with RNA encoding Xcyr61. (J) Activin-treated animal caps derived from embryos injected with RNA encoding Xcyr61 (1,2,3). Note that Xcyr61 constructs do not inhibit induction of muscle by activin. (K-M) Inhibition of Xcyr61 function does not inhibit activin-induced elongation of isolated animal pole regions. (K) Control animal caps: no elongation occurs. (L) Activin-treated animal caps elongate. (M) Animal caps derived from embryos previously injected with antisense morpholino oligonucleotide MO3 undergo elongation. Elongation of such animal caps appears to be more extensive than is observed in control animal pole regions.

View larger version (68K): [in a new window]   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.