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Fig. 8

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ZDB-IMAGE-170222-72
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Figures for Wild et al., 2017
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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 flt1ka601 mutants (dotted box). (c) Endoxifen inducible neuron-specific sflt1 gain-of-function rescues hyper-branching in flt1ka601 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 flt1ka601/+ were used to facilitate biallelic knockout. (fh) Neuron-specific loss of flt1 (flt1ΔNC) 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. (jl) 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 flt1ka601 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 bh,m,n; 25 μm in k,l.

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