Fig. 3

ZBP-89 rescues the hematopoietic but not the vascular phenotype in clo^-/- mutant zebrafish embryos. (A,B) Wild-type whole-mount 3 dpf embryos stained with DAF and examined at 4 X (A) and 10 X (B) magnification, showing blood cells in the heart (red arrow), the dorsal aorta (arrow, B) and the posterior cardinal vein (arrowhead, B). (C,D) Clo^-/- embryo examined at at 4 X (C) and 10 X (D) magnification. No extravascular or circulating blood is seen. Red arrow in C indicates the dilated bloodless heart. (E,F) ZBP-89 sense RNA injected into clo^-/- embryos, shown at two different magnifications. Blood formation in the trunk is evident (arrow in F), but the heart chamber remains dilated and devoid of erythrocytes (E), consistent with the absence of blood vessels. (G,H) Gata1-driven GFP in clo^-/- 48 hpf embryos transgenic (Tg) for gata-1:EGFP, before (G) and after (H) overexpression of ZBP-89. Gata1 is found in the GFP-labeled erythroid lineage (arrow in H) in the anterior ICM of clo^-/- embryos overexpressing ZBP-89. (I-K) Tg (Flk1:EGFP) 48 hpf embryos showing fluorescence in the axial (arrow in I) and intersomitic (short arrow in I) blood vessels; fluorescence in the yolk extension is nonspecific. (J,K) flk1-driven GFP in clo^-/- 48 hpf embryos transgenic for flk1:EGFP before (J) and after (K) overexpression of ZBP-89. Arrows show a lack of blood vessel formation in both cases. (L) ZBP-89 expression profile in two different 18 hpf wild-type (WT1 and WT2) and clo^-/- (clo^-/-1 and clo^-/-2) embryos, assessed using RT-PCR. GAPDH expression was examined simultaneously as a reference.