Zebrafish larvae were injected with vegf-Aa-MO (100 μM), miR-378a-3p mimic (25 μM) or co-injected with either vegf-Aa MO (150 μM) and vegf-Aa zebrafish protein (2.5 nl of 250 ng/ml solution) or miR-378a-3p mimic (25 μM) and a vegf-Aa zebrafish protein (2.5 nl of 250 ng/ml solution) at one to four cell stage. Scale bar = 500 μm.
(A) The phenotype of the larvae was categorized into 4 groups: P1 = no edema, P2 = mild edema, P3 = severe edema, P4 = very severe edema depending on yolk sack. Scale bar = 500 μm. (B) Quantification of loss of circulating high molecular weight proteins by measuring max. eye fluorescence in the retinal vessel plexus of Tg(l-fabp:DBP:EGFP) zebrafish. Scatter plot graph presenting maximum fluorescence intensity of the fish eye was analysed with image J.
(C) Transmission electron microscopy pictures of zebrafish at 120 hpf. Swelling of glomerular endothelium after vegf-Aa-MO injection and thickening of the GBM together with podocyte effacement after miR-378a-3p mimic and after miR-378a-3p mimic/ vegf-Aa protein co-injection is illustrated. Scale bar = 500 nm.
*** p<0.001, n.s. not significant; hpf: hours post fertilisation.