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Fig. 2. Rspo3 regulates the balance between blood and endothelial differentiation. (A-D) Rspo3 is required for blood vessel formation. Xenopus tropicalis embryos were injected equatorially into each blastomere at the four-cell stage with 2.5 ng control morpholino (Co Mo) or Rspo3 Mo as indicated. (A,B) Tadpole morphology; note edema in embryo injected with Rspo3 Mo (B; 69.0%, n=203) but not in control (A) (6.4%, n=173). (C,D) o-Dianisidine erythrocyte staining at tadpole stage. Poor blood vessel formation occurs in the head and erythroid cells accumulate in the ventral region with Rspo3 Mo (D). (E-L) Rspo3 is required for angioblast formation. Xenopus tropicalis four-cell stage embryos were injected equatorially with 2.5 ng Co Mo or Rspo3 Mo, as indicated, into each blastomere and analyzed at tailbud stage by whole-mount in situ hybridization for the indicated genes. (H,L) Inset shows high magnification of the lateral region with Msr-expressing angioblasts. Control and Rspo3 Mo showed Msr staining at 93% (n=75) and 20% (n=87) frequency, respectively. (M-O) Rescue experiment of Rspo3 Mo-induced phenotype. Xenopus tropicalis four-cell stage embryos were injected with 2.5 ng of Co Mo or Rspo3 Mo with or without 50 pg Xenopus Rspo2 mRNA, as indicated, into each ventral blastomere. At gastrula stage (st. 10.5), ventral marginal zones (VMZ) were explanted, cultured until stage 28 and processed for whole-mount in situ hybridization for α-globin. The upregulation ofα -globin expression (23/24) in Rsop3 Mo-injected VMZ and the downregulation of α-globin expression (19/20) in Rspo3 Mo and Rspo2 mRNA-injected VMZ was observed in two independent experiments. (P-W) BSA-, Rspo2- or VEGF-soaked beads (top label) were transplanted in the ventral blood island precursors in neurulae and processed for whole-mount in situ hybridization for the indicated markers (side label) at tadpole stage. Arrowheads indicate ectopic Msr induction. White circles indicate implanted beads. |
