Xenbase Image ID: 76376

Figure 2. R-Spondin 3 Is Required for Xenopus Gastrulation and Noncanonical Wnt Signaling (A) Loss of Rspo3 function causes gastrulation defects in Xenopus embryos. Top, 4-cell stage embryos were micro- injected equatorially into two dorsal blastomeres with 40 ng per embryo of Rspo3 Mo. Note the spina bifida phenotype with two tail ends (arrowheads) in embryos in- jected with Rspo3 Mo (61%, n = 66) but not control (0%, n = 45) embryos. Middle, in situ hybridization for Xbra at gastrula stage (stage 11). Control and Rspo3 Mo embryos showed 100% (n = 20) and 95% (n = 20) normal Xbra stain- ing, respectively. However, note the enlarged blastopore in the Rspo3 Morphant. Bottom, Rspo3 Mo inhibits elon- gation of Activin injected animal caps. (B) Rspo3 signaling requires Sdc4 and Wnt5a to induce gastrulation defects. Embryos were injected into two dorsal blastomeres at 4-cell stage with Morpholinos and/ or mRNA as indicated (40 ng Rspo3 Mo; 100 pg wild- type or dominant negative hSdc4 mRNA; 10 or 20 ng of Sdc4 Mo; 2.5 or 10 ng of Wnt5a Mo [+, and ++]); 250 pg xRspo3 mRNA per embryo were used). (E) Confocal microscopy cell protrusion assay. Dorsal marginal zones at stage 10.5 from embryos injected with membrane-RFP mRNA and 40 ng Rspo3 Mo or 10 ng Wnt5a Mo and/or 50 pg hRspo3 mRNA per embryo were dissociated. White arrowheads indicate protrusions. Right, Quantification of protrusions from three indepen- dent experiments (between 300 and 600 cells counted for each bar). Standard deviation of the mean is indicated. VMZ, ventral marginal zone for control. (F) Top, nuclear phospho-JNK immunostaining in stage 10.5 dorsal mesoderm from embryos dorsally injected with indicated Morpholinos or mRNAs. Bottom, nuclear Hoechst stain. Larger Image Printer Friendly View

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