Effects of Dll4 knockdown on circulation and vascular pattern. (A) Blood flow in intersegmental vessels (ISVs) of wild-type living embryos at 3 dpf. Each line represents a set of ISVs on one side of one embryo, scored for direction of blood flow: upward arrows (red) denote flow from ventral to dorsal; downward arrows (blue), denote flow from dorsal to ventral; dots represent vessels carrying no flow. (B,C) Blood flow in intersegmental vessels of living embryos at 3 dpf injected with either 10 ng MO[Dll4] to knockdown Dll4 (B) or with 10 ng of a 5-base mismatch control morpholino (C). (D-G) fli1:EGFP embryos at 2.5 dpf (D,E) or 5 dpf (F,G) either uninjected (D,F) or injected with 10 ng MO[Dll4] (E,G). White blobs (arrows in D,E) are endothelial cell nuclei. (H) Similar region of a 2.5-dpf dll4-homozygous-mutant embryo (carrying the fli1:EGFP transgene). (I) Endothelial cell counts in the DAs, ISVs and DLAVs of embryos at 3 dpf. Embryos lacking Dll4 or treated with 100 μm DAPT have more cells than uninjected or DMSO-treated control siblings. Both effects are significant at the P=0.001 level (t-test; n≥6 specimens for each treatment; error bars represent s.e.m.). (J) fli1:EGFP embryo at 2.5 dpf injected with 10 ng of a morpholino targeted against casanova (sox32) to block circulation in the trunk. (K) fli1:EGFP-positive dll4-mutant embryo at 2.5 dpf treated from 48 hpf with 2 μM SU5416 to block Vegf signalling. (L) The gut and pectoral fin vasculature of an fli1:EGFP-positive dll4 mutant and a wild-type (normal) sibling embryo, both at 11 dpf. Scale bar: 50 μm in D-H,J,K; 40 μm in L.
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