Figure 2

Figure 2

miR-200c mimic induces edema, proteinuria, podocyte effacement and loss of endothelial fenestrations. Zebrafish were injected with an miR-Ctrl mimic and miR-200c-3p mimic at the one-to-four cell stage. (a) Phenotype pictures of zebrafish larvae at 96 h postfertilization (hpf). Scale bar = 500 µm. (b) Staining the zebrafish pronephroi with hematoxylin–eosin (H&E) at 96 hpf after miR-Ctrl and miR-200c mimic injection in egg stage. Scale bar = 100 µm and 10 µm in the zoom in (c) Quantification of phenotype by severity of pericardial and yolk sac edema in percentage. Examples of phenotypic changes, grading the normal zebrafish phenotype with no edema P1 and the most “edematous” phenotype with P4 at 96 hpf. (d) Timely fusion of the glomeruli at 48 hpf after miR-200c overexpression in Tg (wt1b:EGFP) zebrafish, which express eGFP under the control of the wt1b promotor. Scale bar = 200 µm. (e) Representative images of an eye of Tg(I-fabp:VDBP:EGFP) fish, which were injected with an miR-Ctrl or miR-200c mimic. Scale bar = 150 µm. Quantification of loss of circulating high-molecular-weight proteins by measuring max. eye fluorescence in the retinal vessel plexus of Tg(l-fabp:VDBP:EGFP) zebrafish larvae at 96 hpf. *** p < 0.001. (f) Top: TEM pictures of the zebrafish GFB at 96 hpf after miR-200c mimic and miR-Ctrl injection in egg stage. White arrows show fenestration in the control zebrafish, which are lost in miR-200c-injected zebrafish. Black arrows show podocyte effacement. Scale bar = 500 nm. Bottom: quantification of loss of endothelial fenestrae and podocyte effacement in percentage of representative fish in each group. * p < 0.05; *** p < 0.001.