Fig. 5

Fig. 5 Tgfβ and Vegf Signaling Increases Vascular Permeability and plvapb Expression (A–D) Measurement of hypophyseal permeability using the transgenic biosensor Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax) (A) was performed as described in Figure 4. Larvae were treated with either DMSO (B, n = 14) or TGF-β type I receptor inhibitor, SB431542 (C, n = 12), between 4 and 5 dpf, and extravasation of DBP-EGFP protein was quantified (D, ∗∗p < 0.01; Mann-Whitney test). A.U., arbitrary units. Scale bar: 5 μm. (E–G) The effect of Tgfβ3 on permeability was determined using the triple transgenic system Tg(l-fabp:DBP-EGFP;kdrl:mCherry-caax;pomc:Gal4) (E). The pomc:Gal4 driver was used to generate local hypophyseal clones overexpressing either UAS:tRFP (F, n = 10) or UAS:tgfβ3-2A-tRFP (G, n = 22), as described in Figure 4. Dashed lines mark the border between the permeable hypophyseal capillary and the non-permeable hypophyseal artery. Mosaic expression of Tgfβ3 (G, dotted cell) near the non-permeable artery led to extravasation of DBP-EGFP (inset; white arrows), whereas tRFP expression did not induce that effect (F). Correlation between the co-occurrence of artery extravasation and Tgfβ3 clones was determined by calculating the phi coefficient of association, followed by Fisher’s exact test of association (n = 8/22; p < 0.05) Scale bar: 10 μm. (H–K) Measurement of in situ plvapb expression upon Vegf or Tgf signaling perturbation. Confocal z stack images showing fluorescent in situ hybridization (FISH) of transgenic larvae using probes directed against mRNAs of plvapb. Larvae were treated with either DMSO (H, n = 24), SU5416 (I, n = 19) or SB-431542 (J, n = 6) between 4 and 5 dpf, and plvapb fluorescent intensity was quantified (K, ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; one-way ANOVA). A.U., arbitrary units. Scale bar: 5 μm. Data are presented as mean ± SEM (D and K). See related Figures S4 and S5.