FGF signalling affects NH innervation and vascularisation independently of its effect on AH development and via direct effects on hypothalamic neurons and endothelial cells. Whole-mount immunolabelling (A-D) and confocal in vivo images (E-P) of wild-type (wt) or transgenic fish, as indicated. A-D: lateral views; E-I,M-P: ventral views, anterior to left; J-L: ventral views, anterior to top. (A-D) Double-labelled specimens for AcTub and Prolactin. (A,B) Maximal confocal projections at 72 hpf (A,B) or 120 hpf (C,D) in wt or after heatshock-activated dnFgfr1 expression at 33 hpf (B) or 42 hpf (D). Prolactin expression in the AH is normal, whereas NH axons appear to stall (compare B with Fig. 5I, and D with Fig. 5J). (E-I) Maximal projections of kdrl-driven mCherry fluorescence in endothelial cells at 72 hpf (E-G) or 75 hpf (H,I). Heatshock-activated dnFgfr1 expression at 33 hpf and 48 hpf leads to loss of hypophyseal artery and capillaries, but the hypophyseal vein remains present (compare panel G with E and with Fig. 6T). Single heatshock application at 33 hpf leads to an intermediate vessel phenotype (F), whereas heatshock applications at 48 hpf and 60 hpf leave hypophyseal vascularisation unaffected (compare panel I with H; inset in I shows strong and widespread expression of dnFgfr1-GFP). (J-N) Injection of UAS:dnfgfr1-mcherry plasmid (J,K,M) or UAS:mCherry plasmid (L,N) into tg(otpb:egfp);tg(avlp:gal4) double transgenic animals leads to mosaic expression of dominant-negative Fgfr1-mCherry fusion protein (J,K) or mCherry protein (L) in a subset of pre-optic hypothalamic cells (marked by otpb-driven EGFP expression, green). Avpl+ neurons expressing dnFgfr1-mCherry (arrowheads in J,K) fail to project axons and to innervate the NH (M), whereas neurons lacking dnFgfr1-mCherry display normal axogenesis (J,K). Axogenesis (L) and NH innervation (N) are also normal in Avpl+ neurons of control transgenics expressing mCherry (arrows in L). (O,P) Injection of UAS:dnfgfr1-egfp plasmid into tg(fli1:gal4);tg(kdrl:mcherry) double transgenic animals leads to mosaic expression of dominant-negative Fgfr1-EGFP fusion protein (green) in a subset of endothelial cells (marked by kdrl-driven mCherry expression, red). Contribution of cells expressing dnFgfr1 to one of the hypophyseal capillaries abrogates its fusion with the hypophyseal vein (O; arrowhead), whereas the fusion is not affected by cells carrying a UAS.egfp control plasmid (instead of UAS:dnfgfr1-egfp) (P; arrows). Arrow in O indicates normally wired non-transgenic endothelial cells. cadi, caudal division of internal carotid artery; hya, hypophyseal artery; hyc; hypophyseal capillary; hyv, hypophyseal vein; nh, neurohypophysis; phs, primary head sinus. Scale bars: 50 μm in A-I,M-P; 15 μm in J-L.