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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. |
