Vegfd modulates SoxF driven gene expression in vivo in zebrafish. (A) Confocal projection of Tg(−6.5kdrl:EGFP; fli1a.ep:DsRedEx), in which the EGFP transgene is driven by a 6.5-kb kdrl promoter fragment. The expression of EGFP but not DsRed was selectively reduced in single morphants and appeared absent in Sox18/Sox7 dKD embryos. (B) In situ hybridization for EGFP transcript confirmed downregulation in Sox18/Sox7 dKD. (C-D) qRT-PCR showed a mild reduction of endogenous kdrl mRNA concentration (C) in dKD embryos, whereas EGFP expression levels were dramatically decreased (D) (relative to cdh5). (E) Map of the 6.5-kb kdrl promoter fragment showing the position of SOX responsive elements (S1, S2) within the −4.3 to −3.5 kb minimal promoter region previously identified as sufficient to drive EGFP expression.⁵³  The 6.3-kb Δsox promoter fragment was generated by excising approximately 200 bp that includes S1 and S2. Δkdrl promoter was generated by removing a 6.3-kb fragment and was used as a negative control to assess EGFP expression. (F) In confocal projection of wild-type zebrafish injected with different kdrl:EGFP constructs, the level of EGFP expression in injected embryos was scored qualitatively (G), and quantified by RT-PCR (H) (relative to cdh5). Loss of the 200-bp fragment containing S1 and S2 led to a complete loss of EGFP expression. (I) Fluorescence polarization assay showing the direct binding of SOX18 protein to the SOX response elements in vitro (S1 black and S2 gray). The 100 times excess nonlabeled wild-type DNA probes were sufficient to displace 100% of the fluorescent-tagged probes, whereas the same excess of mutated probe failed to efficiently compete (ANOVA, post hoc Dunnett's test, multiple comparison with DNA free controls, ± standard deviation, n = 3). (J) Expression level of EGFP in the transgenic line Tg(−6.5kdrl:EGFP) was assessed by qRT-PCR. Knock down of Vegfd alone influenced EGFP transcript (23% reduction). dKD of either Sox18/Vegfd or Sox7/Vegfd shows a further reduction in EGFP expression. The data are shown as the mean ± standard error of the mean of 3 to 6 independent experiments. ****P < .0001, ***P < .001, *P < .05, ns: nonsignificant by 1-way ANOVA test. Red dashed line indicates a proposed phenotypic threshold (see the “Discussion” section). Scale bars represent 100 μm (A), 200 μm (B), and 100 μm (F).