Fig. 4

Fig. 4 Vegf Signaling Regulates Hypophyseal Capillary Morphogenesis and Permeability (A–D) Morphological analysis and cell count of the hypophyseal artery and capillary loop and vein using the Tg(fli1:nuc-EGFP) line, which labels endothelial cell nuclei (A). The transgenic line Tg(hsp70l:sFlt1) was used to inhibit Vegf/Vegfr2 signaling temporally. Control (B, n = 10) and Tg(hsp70l:sFlt1) larvae (C, n = 15) were subjected to multiple 28οC to 37οC temperature shifts between 3 dpf and 5 dpf. The capillary loop cell number (D) was significantly higher in Tg(hsp70l:sflt1) larvae (∗∗p < 0.01; Student’s t test). Correlation between the co-occurrence of abnormal loops and temporally induced sFlt1 expression was determined by calculating the phi coefficient of association, followed by Fisher’s exact test (n = 6/15; p < 0.03). Scale bars: 10 μm. (E–H) Hypophyseal clones (E) overexpressing control tRFP (F) or the soluble antagonist sFlt1 (E and G). Double transgenic Tg(pomc:Gal4;fli1:nuc-EGFP) embryos were injected with transposon-based transgenic expression vectors containing either control UAS:tRFP (n = 19) or UAS:mCherry-2A-sFlt1 (n = 15), allowing for mosaic gene expression in discrete hypophyseal cells (magenta). Cell numbers in the capillary loop (H) were significantly higher in the mutant (∗p < 0.05; Mann-Whitney test). The schemata shown at the bottom of panels depict the normal (F) and abnormal (G) connectivity of the hypophyseal capillary loop in 5 dpf larvae with mosaic expression. Correlation between the co-occurrence of the abnormal loop and sFlt1 clones was determined by calculating the phi coefficient of association, followed by Fisher’s exact test (n = 5/15; p < 0.05). Scale bars: 5 μm. (I–M) Assessment of the effect of Vegfr inhibitor, SU5416, on permeability in transgenic reporter Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) larvae. D-vitamin binding protein (DBP) expressed in hepatocytes (l-fabp promoter) served as an endogenous permeability biosensor (I). Extravasation of the DBP-EGFP protein from the permeable neurohypophyseal vascular cells (J) was measured in the area inside the hypophyseal capillary loop (dashed line in K). Larvae were treated with DMSO (K, n = 9) or SU5416 at 5 μM (L, n = 13) between 4 and 5 dpf. Treatment with SU5416 led to reduced extravasation of DBP-EGFP from the hypophyseal loop (M, ∗p < 0.05; Mann-Whitney test; for each treatment). A.U., arbitrary units. Scale bar: 5 μm. Data are presented as mean ± SEM (D, H, and M). See related Figures S4 and S5.