Knockdown of crlra in combination with VEGF inhibition prevents arterial differentiation in ptc1;ptc2 mutants. (A) Wild-type somitic (green arrowhead) and DA crlra expression (red arrowhead). (B) Increased somitic (green arrowhead) and DA (red arrowhead) crlra expression in ptc1;ptc2 embryos. (C) Normal somitic crlra expression in uninjected wild-type non–double mutant embryos (arrowhead; n = 58 of 66). (D) Decreased somitic crlra expression in non–double mutant crlra morphants (n = 58 of 71). (E-F) No difference in crlra expression (arrowheads) was detected between uninjected (n = 5 of 66) and crlra morphant (n = 4 of 71) ptc1;ptc2 embryos. Uninjected wild-type siblings treated with SU5416 from tailbud until collection exhibited total absence of vascular ephrinb2a expression (K red arrowhead; n = 40 of 50) compared with controls (G red arrowhead; n = 46 of 49). SU5416-treated ptc1;ptc2 mutants showed increased ephrinb2a (L red arrowhead) and ectopic expression in PCV region (L blue arrowhead; n = 4 of 50), as did uninjected ptc1;ptc2 mutants (H red and blue arrowheads; n = 3 of 49). Uninjected SU5416-treated ptc1;ptc2 embryos exhibited no vessel sprouting compared with DMSO-treated ptc1;ptc2 embryos. ephrinb2a expression was down-regulated in the DA of non–double mutant crlra morphants treated with DMSO (I red arrowhead; n = 78 of 96), whereas non–double mutant crlra morphants treated with SU5416 exhibited a loss of vascular ephrinb2a expression (M red arrowhead; n = 165 of 183). ptc1;ptc2 crlra morphants treated with DMSO showed strong ephrinb2a expression (J red arrowhead), which was present ectopically in the region of the PCV (J blue arrowhead; n = 8 of 96), whereas vascular ephrinb2a was absent in ptc1;ptc2 crlra morphants treated with SU5416 (N red arrowhead; n = 10 of 186). (O) Proposed model for arterial differentiation.