Fig. 8

Neuronal Flt1 regulates vascular branching by titrating neuronal Vegfaa.

(a) Schematic representation of endoxifen inducible gain-of-function approach in zebrafish. In the present situation Gal4 is under the control of neuron-specific promoters elavl or XIa.Tubb. Expression can be observed within 1.5 h upon endoxifen application. (b) Hyper-branching in flt1^ka601 mutants (dotted box). (c) Endoxifen inducible neuron-specific sflt1 gain-of-function rescues hyper-branching in flt1^ka601 mutants; compare dotted box in c and b. Purple arrowheads indicate vISVs; endoxifen was applied at 52 hpf. (d) Quantification of rescue in (b,c), mean±s.e.m, n=15-19 embryos per group. (e) Approach for generating a neuron-specific flt1 mutant. Cas9 was expressed under control of neuronal promoter Xla.Tubb; sgRNA was expressed ubiquitously, resulting in Cas9 activity in neuronal cells only (domain marked by orange border). Heterzygous flt1^ka601/+ were used to facilitate biallelic knockout. (f–h) Neuron-specific loss of flt1 (flt1^ΔNC) induces ectopic sprouting (h), sprouts in yellow dotted ellipse, arrowheads indicate neuronal cells with Cas9 expression. (i) Quantification of ectopic sprouting for indicated genotypes. Note that neuron-specific loss of flt1 significantly augments ectopic sprouting (green bar) mean±s.e.m, n=16 embryos per group, t-test. (j–l) Transplantation of flt1 mutant neuronal cells (k) and endothelial cells (l) into WT. Note: transplantation of flt1 mutant neuronal cells induced sprouting (k, arrowheads); 9 out of 12 neuronal cell transplantations resulted in sprout formation. In all 10 endothelial cell transplantations, sprouts were absent (l). (m,n) Low dose morpholino-mediated reduction of vegfaa expression in flt1^ka601 mutants rescues sprouting defects; compare dotted box in (m,n). (o) Quantification of rescue in (m,n), mean±s.e.m., n>5 per group, t-test. DA, dorsal aorta; PCV, posterior cardinal vein; DLAV, dorsal longitudinal anastomotic vessel; NT, neural tube. GOI, gene of interest; POI, protein of interest; iNC, inducible, neuronal cell specific gain-of-function; ΔNC, neuron-specific loss of flt1; MO, morpholino. Scale bar, 50 μm in b–h,m,n; 25 μm in k,l.