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Fig. 6 Chemical inhibition of Vegf family receptors phenocopies vegfc loss-of-function. (A) Experimental schematic showing SU5416 dosage starting at 32 hpf, followed by time-lapse imaging from 48 to 52 hpf. The hatched area indicates the time-lapse window, and the gray area indicates the time of SU5416 treatment. (B) Treatment of embryos with SU5416 created high levels of autofluorescence in the yolk sac. Therefore, we present images with levels (brightness and contrast) optimized for GFP signal in DMSO-treated control embryos (left column) and SU5416-treated embryos (right column). Single frames of the time-lapse (hpf:min) show cd41:gfp+ HSPCs (green) budding from the flk:mCherry+ DA (magenta) (Movie 9). White arrows indicate cells undergoing EHT. Compared with DMSO controls (top panels), there are very few budding HSPCs in SU5416-treated embryos (bottom panels) (Movie 10). Dotted lines outline pronephric tubules. (C) Quantification of the total number of cd41:gfp+ HSPCs budding from the DA between 48 and 52 hpf. The total number of events observed are divided by the duration of the experiment (4 h) to give the number of events per hour. The data shown is from one experiment with n=5 DMSO- and n=5 SU5416-treated embryos. P=0.0002 (unpaired two-tailed t-test). (D) In vivo imaging of the CHT in cd41:gfp/flk:mCherry embryos at 72 hpf, after treatment with SU5416 from 32 hpf, shows a reduction in HSPC numbers. (E) Quantification of cd41:gfp+ cells within the CHT, as shown in D; DMSO control (n=11) versus SU5416-treated (n=12). P=0.0001 (unpaired two-tailed t-test). (F) Treatment with SU5416 increases the percentage of embryos with high cmyb expression compared with DMSO-treated embryos. cmyb expression was scored in the CHT at 72 hpf. (G) Percentage of total embryos scored and classified as having low or high cmyb expression, P=6.73×10−6 (chi-square goodness of fit test). (H) Treatment with SU5416 increases the percentage of embryos with high mpx expression compared with DMSO controls. (I) Percentage of total embryos scored and classified as having low or high mpx expression, P=4.06×10−8 (chi-square goodness of fit test). SU5416 dose is 0.75 µM in DMSO. Scale bars: 50 µm (B,D).