Influence of genetic background on the proteinuria phenotype. (A) Tg(l-fabp:eGFP-DBP) zebrafish backcrossed on homozygous for either the AB (AB fish) or the nacre (nac^w2) background (nacre fish) as well as heterozygous zebrafish for nacre and AB background were treated with PAN at 46?h post-fertilization (hpf). Graph depicts max. fluorescence of circulating eGFP-DBP in the retinal vessel plexus of the fish eye 96?hpf. (B) Representative images of phenotypes of Tg(l-fabp:eGFP-DBP) zebrafish backcrossed on homozygous for either the AB (AB fish) or the nacre (nac^w2) background (nacre fish) were treated with PAN (4?mg/ml) or DMSO at 46?hpf as indicated. Pictures were taken at 96?hpf. (C) Representative transmission electron microscopy images of the glomerular filtration barrier of Tg(l-fabp:eGFP-DBP) zebrafish backcrossed on homozygous for either the AB or the nacre (nac^w2) background were treated with PAN (4?mg/ml) or DMSO at 46?hpf as indicated. Fish were collected at 120?hpf. Podocyte effacement after PAN treatment was more severe in nacre fish compared to AB fish. ***P<0.001, n.s., non-significant. Scale bars: (B) 500 μm, (C) 500 nm.

Mitf knockdown model and PAN treatment. (A) Phenotype pictures of Tg(l-fabp:eGFP-DBP) zebrafish backcrossed on AB background that were injected with either a mitf MO or a scrambled MO in a concentration of 250?µM in one to four-cell stage. Pictures were taken at 96?hpf. (B) Tg(l-fabp:eGFP-DBP) zebrafish backcrossed on AB background were injected with a mitf MO or a scrambled MO in a concentration of 250?µM in one to four-cell stage. At 46?hpf fish were treated either with PAN (4?mg/ml) or DMSO. Intensity of circulating eGFP-DBP (arbitrary units) in the retinal vessel plexus of the fish eye was measured at 96?hpf. Representative images of the fluorescence of circulating eGFP-DBP in the retinal vessel plexus of the fish eye 96?hpf are shown at the bottom. n=25–28 per group. (C) Q-PCR for inf2 mRNA expression in whole zebrafish at 120?hpf. Zebrafish (AB strain) were injected with CTRL-MO or a mitf-MO at one to four-cell stage. Data from three different experiments. Significance was tested with two-way ANOVA: **P<0.01, ****P<0.001, ns, non-significant. Scale bars: 500?µm.