Fig. 6. (A) Flk1-dependent Vegfa induction of Pax2. The injection is noted at the top of each panel, and the antibody treatment and hybridization probes are noted at the bottom of each panel. Gfp (a) or Vegfa (b-e) expression vectors were injected at the edge of the condensed mesenchyme. Rat IgG (d) or Flk1-blocking antibody (e) were added at the time of injection. Six hours after injection, cultures were analyzed by in situ hybridization with Pax2 antisense (a,b,d,e) or sense (c) probe. (B) Flk1-dependent Pax2 induction of Gdnf expression. Panels are labeled as in A. Pax2 expression (a,b.d,f) or control (c,d,g,h) vectors were injected and Gdnf antisense probe was used in all panels. (C) Real-time PCR analysis of Pax2 mRNA expression level in organ cultures after Flk1 blockade or treatment with actinomycin-D. Treatments are shown in the figure box. SU1498, actinomycin-D, SU1498 with actinomycin-D or DMSO, were added to the culture medium at the beginning of the culture period, (time 0). At 0, 1.5, 3, 4.5, 6, or 7.5 hours after beginning cultures, real-time PCR was performed to measure levels of Pax2 mRNA. All Pax2 Ct values were normalized to the Gapdh Ct values and converted to relative mRNA levels as described in the methods. Relative RNA levels are shown on a log scale. (D) Flk1 dependency of Pax2 expression. Panels a-h were injected with the Pax2 expression vector. The hybridization probes are noted at the bottom of each panel (a-d), and the antibody treatment is noted at the top (a-d) or bottom (e-h) of each panel. (a,e) Rat IgG; (b,f) Flk1-blocking antibody; (c,g) DMSO 1:1000; (d,h) SU1498 treatment. (a-d) In situ hybridization with Pax2 antisense probe; (e-h) staining with anti-Pax2 antibody. Pax2 mRNA or protein expression in response to injection of the Pax2 expression vector could be blocked by antibody or pharmacological inhibition of signaling through Flk1. (i-l) Injections of the GFP expression vector, detecting direct GFP fluorescence.