Fig. 7

Neurons are a major source of Vegfaa and attract sprouting vessels.

(a) FACS procedure for obtaining neuronal cells in control and vhl morphants using Tg(Xla.Tubb:DsRed)^zf148 neuronal reporter embryos. (b) Quantification of vegfaa expression using real-time qPCR in FAC-sorted cell populations at 3 dpf. Note that neuronal cells expressed significantly more vegfaa than non-neuronal cells. Loss of vhl promoted neuronal vegfaa expression. Mean±s.e.m., n=3 separate experiments in triplicate (two-way ANOVA). (c) Schematic representation: loss of vhl augments vegfaa transcription, loss of flt1 augments Vegfaa bioavailability; combining both mutants augments Vegfaa bioavailability above single mutant level. (d) Trunk vasculature in flt1^ka601;vhl^hu2114 double mutants at 4 dpf. Note the severe hyper-branching at the level of the neural tube, red-dotted box. (e) Schematic representation of optical section (shown in f–h) through the neural tube and associated trunk vasculature. (f–h) Dorsal view on optical section through WT (f), flt1^ka601 (g) and flt1^ka601;vhl^hu2114 double mutants (h). Note invasion of sprouts into the neural tube in double mutants (arrowheads in h). Red circle indicates position of ISVs, dotted line neural tube boundary. (i) Transverse 3D-rendered view of vasculature (green) through the trunk in WT (left panel) and flt1^ka601;vhl^hu2114 double mutants (right panel); note vessels penetrating the neural tube in mutant (compare vessel in dotted circle right panel, arrowhead; such vessels are absent in WT left panel; representative image from 3 separate experiments). (j) Representative image of spinal cord vascular network in Tg(xIa.Tubb:DsRed)^zf148; Tg(kdrl:EGFP)^s843 double transgenic at 13 dpf. (k,l) Comparison of trunk vasculature in WT at 4 dpf (k) and at 13 dpf (l); note the emergence of ectopic branches (pink arrowheads) at level of the spinal cord. DA, dorsal aorta; f.c. fold change; KD, knockdown; NT, neural tube; NC, neuronal cell; PCV, posterior cardinal vein. Mutants are in Tg(kdrl:EGFP)^s843 background. Scale bar, 50 μm in d; 25 μm in f–l.