Fig. S8

Targeting neuronal and vascular Flt1 and Vegfaa

(a) Vascular-specific depletion of flt1 (flt1^ΔEC) in flt1^ka601/+ heterozygous animals does not induce ectopic venous sprouting. Embryos are in Tg(kdrl:has.HRAS-mcherry)^s916 background. (b) Graphical illustration of multiplex miRNA construct with GFP or DsRed reporter, using miR-155 backbone coupled to three custom made miRNAs directed against the 3’UTR of sflt1. (c) Neuron-specific targeting of sflt1 with miRNA approach induced ectopic venous sprouting in WT. 9 out of 12 vISVs with high miRNA expression in the adjacent neuronal cells (in green) formed sprouts. (d) Vascular-specific targeting of sflt1 with miRNA approach failed to induce ectopic sprouting. vISVs expressing the miRNA construct (in red) did not induce sprout formation n=21. (e,f) Endoxifen inducible neuron specific vegfaa165 gain-of-function induced at 52hpf after AV remodeling induces hyper-branching (arrowheads). (g) Endoxifen inducible neuron specific vegfaa121 gain-of-function induced sprouting (arrowhead). (h) Endoxifen inducible neuron specific vegfc gain-of-function (yellow arrowheads) induced at 52hpf does not induce ectopic sprouting at level of neural tube. (i,j) Endoxifen inducible neuron specific vegfaa165 gain-of-function induced at 30hpf, before completion of AV differentiation induces severe thickening of ISVs and abnormal vascular remodeling; compare dotted box in (i) and (j). (k) Constitutive (non-inducible) neuron-specific sflt1 gain-of-function blocked ISV development; most ISVs were missing (arrowheads). (l-n) Flt1^ka601 mutant treated with DMSO (l); treated with Kdrl receptor signaling inhibitor ki8751 (m, R2 inhibitor); treated with Flt4 tyrosine kinase inhibitor MAZ51 (n, R3 inhibitor). (o) Quantification of l-n. Mean ± s.e.m, n=11/group, t-test. iNC, inducible, neuronal cell specific gain-of-function; R2, VEGF receptor 2; R3, VEGF receptor 3. miR, microRNA. Scale bar, 25μm in a, c-n.