Fig. 4. Vegf mediates R-spondin/Wnt signaling during hematopoiesis. (A) RT-PCR analysis of ventral marginal zones (VMZ), experimental set up as in Fig. 3A-C. (B) Four-cell stage embryos were injected with 10 ng Co Mo or VEGF Mo, as indicated, into both ventral blastomeres. Beads soaked in BSA or Rspo2 were implanted at neurula stage (white circles), and processed for whole-mount in situ hybridization for -globin at tadpole stage. (C) Experimental design for experiment in D. Top: `inducer' embryos were injected with 100 pg Wnt3a or 500 pg Rspo2C (well-secreted variant) mRNA plus lineage tracer FLDx (fluorescence lysinated dextran), cultured until stage 8 and treated with cycloheximide (CHX, 40 µg/ml) for 30 minutes. Lower panel: `responder' uninjected animal caps were explanted at stage 8, cultured until control siblings reached stage 15, and treated with or without CHX (10 µg/ml) for 30 minutes. Responder animal caps were bissected, sandwiched with or without animal cap of inducer embryos and cultivated for another 3 hours to permit induction in the presence of CHX (10 µg/ml). Responder animal tissues were harvested and analyzed by RT-PCR shown in D. (D) Vegf induction is an immediate early response to Wnt/R-spondin signaling. Uninjected animal caps (responders) were co-cultured with fluorescently labeled animal caps injected with Wnt3a and Rspo2 mRNA (inducers) as shown in C. Responder animal caps were cultured until control siblings reached stage 15 and treated with or without cycloheximide (CHX) for 3 hours. Responder animal caps were harvested separately and analyzed by RT-PCR for the markers indicated. As a control for the efficacy of CHX treatment, the (indirect) inhibition of keratin expression by Wnt3a and Rspo2 (lanes 2-3) is blocked by CHX (lanes 4-9).