Fig. 6

VEGF signaling is dispensable for induction but necessary for maturation of Etv2 induced vasculature.

(A–D) Control kdrl:GFP embryos (A) or kdrl:GFP/hsp70l:etv2 (B–D) embryos following heat shock at 24 hpf and imaged at 24 h or 36 h post–heat shock. Control embryos exhibit normal vascular kdrl:GFP expression (A), while kdrl:GFP/hsp70l:etv2 embryos exhibit the ectopic GFP and morphological changes previously described (B–D). (E–H) VEGFAa morpholino (VEGF-MO) treated embryos lack intersomitic vessels (E) but still induce kdrl:GFP in muscle fibers (F). However, kdrl:GFP⁺ muscle fibers do not undergo the normally observed morphological changes following heat shock–induced expression of Etv2 (G,H). (I–L) Overexpression of VEGFAa¹²¹ (VEGF OE) driven by the hsp70l promoter results in disorganization and expansion of the normal vasculature (I). Following heat shock–induced expression of Etv2, no significant change in the number of muscle fibers expressing kdrl:GFP is observed (J). However, the morphological changes observed are accelerated in the presence of elevated VEGF (K,L). (M–P) Treatment of embryos with SU5416, a Kdr inhibitor, similarly inhibits intersomitic vessel development in control embryos (M). However, drug treatment does not inhibit induction of kdrl:GFP following heat shock–induced Etv2 expression in muscle (N). The morphology and survival of these fibers is compromised when Kdr is inhibited (O,P). (Q–V) Removal of VEGF inhibitor SU5416 24 h following heat shock allows for survival and maturation of transdifferentiated cells. Kdrl:GFP/hsp07l:etv2 embryos were heat shocked at 22 hpf and then treated with DMSO carrier or SU5416 for 24 h at which point the drug was either maintained (S,T) or removed (U,V) and the embryos were allowed to develop until 72 hpf. (Q,R) DMSO controls exhibit a transdifferentiated vascular network similar to that in untreated controls. (S,T) Sustained SU5416 treatment largely abolishes the kdrl:GFP⁺ vascular network. (U,V) Removal of SU5416 24 h post–heat shock results in the development of a vascular network similar to controls (Q,R), suggesting VEGF signaling modulates the survival and maturation of muscle-derived vessels and not the initial induction. For all experiments at least 20 embryos were observed with similar results.