Notch-independent Alk3a/b signalling is involved in venous identity in zebrafish. a Loss of Notch signalling had no effect on the expression of the venous Ephb4-2:GFP transgene in tg(Ephb4-2:GFP) transgenic zebrafish. Representative 48 hpf embryos demonstrate similar intensities of vein-specific green fluorescent protein (GFP) expression in both control and DAPM-treated embryos. Red bracket = dorsal aorta, white bracket = posterior cardinal and ventral vein. Graph depicts observed expression pattern of GFP in tg(Ephb4-2:GFP) embryos for control (n = 51) and 100 µM DAPM-treated embryos (n = 57); black denotes high expression, grey denotes weak. See Supplementary Fig. 12 for Coup-TFII-965:GFP results and controls. b Morpholino (MO)-induced reduction of alk1 and alk2 had little effect on Ephb4-2:GFP expression in 48hpf tg(Ephb4-2:GFP) transgenic zebrafish, whereas reduction of alk3a/b resulted in significantly decreased transgene expression. Representative 48 hpf tg(Ephb4-2:GFP) embryos demonstrate reduced GFP expression after alk3a/b MO injection. Red bracket = dorsal aorta, white bracket = posterior cardinal and ventral vein. Graph depicts observed expression patterns of GFP for control (n = 51), alk1 MO (n = 45), alk2 MO (n = 45) and alk3a/b MO (n = 48); black denotes high expression, grey denotes weak expression and white denotes no detectable GFP expression. c Whole-mount in situ hybridization for bone morphogenetic protein (BMP) receptors alk3a and alk3b compared to pan-endothelial kdrl in wild-type zebrafish embryos at 20, 24, 28 and 36 hpf. Earlier time points can be seen in Supplementary Fig. 13. Both alk3a and alk3b were detected in the axial vein but not in the artery, with greater early expression seen for alk3b. Red bracket = axial artery, blue bracket = axial vein. d Whole-mount in situ hybridization for BMP ligands bmp2b and bmp4compared to pan-endothelial kdrl in wild-type zebrafish embryos at 28 hpf. Both bmp2b and bmp4 showed stronger expression around the axial veins comparative to the dorsal artery. Red bracket = axial artery, blue bracket = axial vein. All scale bars represent 100 μm. See also Supplementary Figures 12–13
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