Fig. 3

Characterization of nrp1a^sa1485 mutant fish. (A) Structure of Nrp1a in wild-type (nrp1a^+/+) (left) and nrp1a^sa1485 mutant fish (right). The point mutation results in the generation of a premature stop codon at aa 206, resulting in a truncated Nrp1a fragment. Blue diamonds indicate CUB (a1 and a2) domains, orange circles indicate FA58C (b1 and b2) domains, the green square indicates the MAM domain and the brown square indicates the C-terminal domain. (B) Sequencing chromatograms of wild-type fish, heterozygous nrp1a^sa1485/+ and homozygous nrp1a^{sa1485/sa1485} mutant fish. An early stop codon (nonsense mutation) TAA, replaces the wild-type TAC codon at aa 206. The genotypes of 14 zebrafish embryos 48 h post fertilization (hpf) were compared against the expected Mendelian ratio after heterozygous fish incross. (C) Absolute RT-qPCR of wild-type (WT; black circles, white bar) or nrp1a^sa1485 homozygous mutant (black squares, gray bar) uninjured adult zebrafish hearts under basal conditions. nrp1aexpression was significantly decreased in nrp1a^sa1485 samples, suggesting nonsense-mediating decay. ****P<0.0001 (two-tailed t-test; n=7 with each n being a pool of three ventricles). Data are means of normalized copy numbers per reaction±s.e.m. (D) Nrp1a antisense (AS) in situhybridization of wild-type (upper row) or nrp1a^sa1485 homozygous mutant (lower row) embryos 24 hpf. Nrp1a expression was clearly decreased in nrp1a^sa1485 samples. Scale bars: 50 μm. (E) Western blot of wild-type (WT) or nrp1a^sa1485 homozygous mutant uninjured adult zebrafish ventricle lysates (top). Lysates were immunoblotted with an antibody targeting the Nrp1 cytoplasmic domain and Gapdh. Note the absence of C terminus detection of Nrp1a in the nrp1a^sa1485 samples. Western blot quantification of Nrp1a (plain bars, circles for wild type, squares for nrp1a^sa1485) and Nrp1b (striated bars, upward triangles for wild type, downward triangles for nrp1a^sa1485) normalized to Gapdh (one-way ANOVA with Sidak's post hoc test for multiple comparisons of n=4), confirming the significant reduction in Nrp1a expression (****P<0.0001; plain white bar for wild type versus plain gray bar for nrp1a^sa1485), whereas Nrp1b was not significantly different between wild type (white striated bar) and nrp1a^sa1485 (gray striated bar) (P=0.219) (bottom).