Vegf-Aa knockdown by vegf-Aa-MO or miR-26a-5p mimic leads to edema and loss of plasma proteins. Zebrafish were either injected with control morpholino (CTRL-MO), a vegf-Aa morpholino (vegf-Aa-MO), a vegf-Ab morpholino (vegf-Ab-MO), a control miR mimic (miR-CTRL) or a miR-26a-5p mimic (miR-26a-5p) alone (−) or as co-injection with a recombinant zebrafish Vegf-Aa protein (Vegf-Aa) (+) at one to four cell stages. *p <  0.05, **p <  0.01, ***p < 0.001, n.s. not significant. (A) Phenotype pictures of zebrafish larvae at 96 hpf. Scale bar = 500 µM. (B) Quantification of edemous phenotype of zebrafish larvae at 96 hpf. The edemous phenotype was categorized in 4 groups: P1 = no edema, P2 = mild edema, P3 = severe edema, P4 = very severe edema, Dead fish are those found dead between 72 hpf and 96 hpf; CTRL-MO n = 15, vegf-Aa-MO n = 20, vegf-Aa-MO + Vegf-Aa n = 21, vegf-Ab-MO n = 16, vegf-Ab-MO + Vegf-Aa n = 16, miR-CTRL n = 16, miR-26a-5p n = 14, miR-26a-5p + Vegf-Aa n = 24, WT + Vegf-Aa n = 17, WT n = 18, total n = 177. (C) TaqMan qPCR for miR-26a-5p in whole zebrafish larvae lysate 120 h after injection of a miR-26a-5p mimic (miR-26a-5p) or a miR control (miR-CTRL) in one to four cell stage. ***p < 0.001. n = 12 for each group, total n = 36. (D) Survival of zebrafish over time. Survival is given in percent [%] of zebrafish of each group. a) Survival of zebrafish injected with a vegf-Aa morpholino (vegf-Aa-MO) in black versus co-injection of a vegf-Aa morpholino and a Vegf-Aa protein (vegf-Aa-MO + Vegf-Aa protein) in red; n = 64. b) Survival of zebrafish injected with a vegf-Ab morpholino (vegf-Ab-MO) in black versus co-injection of a vegf-Ab morpholino and a Vegf-Aa protein (vegf-Ab-MO + Vegf-Aa protein) in red; n = 62. c) Survival of zebrafish injected with a miR-26a-5p mimic (miR-26a-5p) in black versus co-injection of a miR-26a-5p mimic and a Vegf-Aa protein (miR-26a-5p + Vegf-Aa protein) in red; n = 69.

Vegf-Aa knockdown by vegf-Aa-MO or miR-26a-5p mimic causes loss of plasma proteins in zebrafish larvae. Zebrafish were either injected with control morpholino (CTRL-MO), a vegf-Aa morpholino (vegf-Aa-MO), a vegf-Ab morpholino (vegf-Ab-MO), a control miR mimic (miR-CTRL) or a miR-26a-5p mimic (miR-26a-5p) alone (−) or as co-injection with a recombinant zebrafish Vegf-Aa protein (Vegf-Aa) (+) at one to four cell stages. (A) Representative images of eye of an unaffected Tg(l-fabp:DBP:eGFP) transgenic fish appears green due to the fluorescent plasma protein in the retina vessels (left). In fish with proteinuria, the eye appears dark due to loss of green fluorescent plasma proteins (right). (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 larvae at 96 hpf.***p < 0.001, n.s. not significant; CTRL-MO n = 15, vegf-Aa-MO n = 20, vegf-Aa-MO + Vegf-Aa n = 21, vegf-Ab-MO n = 16, vegf-Ab-MO + Vegf-Aa n = 16, miR-CTRL n = 16, miR-26a-5p n = 14, miR-26a-5p + Vegf-Aa n = 24, WT + Vegf-Aa n = 17, WT n = 18, total n = 177.

Vegf-Aa knockdown by vegf-Aa-MO or miR-26a-5p mimic cause impairments in intersegmental tail vessel sprouting. (A) Western blot for Vegf-Aa and normalization with Gapdh of whole zebrafish larvae lysate (10 µg of a pool of 10 fish). Zebrafish were injected with either a recombinant zebrafish Vegf-Aa protein (Vegf-Aa protein), a control morpholino (CTRL-MO), a vegf-Aa morpholino (vegf-Aa-MO), a control miR mimic (miR-CTRL) or a miR-26a-5p mimic (miR-26a) at the one to four cell stages. Zebrafish were harvested at 96 hpf. Quantification of western blot bands are shown below the blot. n = 12 fish per group, total n = 48. Full-length blots are presented in supplementary Fig. 3. (B) Phenotype pictures of zebrafish show blood pooling in tail region (arrow heads) in zebrafish that were injected with a vegf-Aa morpholino (vegf-Aa-MO) or a miR-26a-5p mimic (miR-26a-5p) were as control injected fish (CTRL-MO and miR-CTRL) appeared normal. Scale bar: 4 mM. (C) Pictures of tail vessel region of Tg(l-fabp:DBP-eGFP/flk-mcherry) zebrafish. These transgenic fish express a red fluorescent endothelium and green fluorescent plasma protein. Zebrafish were injected with either a control morpholino (CTRL-MO), a vegf-Aa morpholino (vegf-Aa-MO) a miR-26a-5p mimic (miR-26a-5p), a control miR mimic (miR-CTRL) alone (−) or together with a recombinant zebrafish Vegf-Aa protein (Vegf-Aa) (+) at the one to four cell stages. Vessel pictures were taken at 96 hpf. Arrow heads illustrate defects in sprouting of intersegmental vessel in vegf-Aa-MO and miR-26a-5p mimic injected fish. DLAV: dorsal lateral anatomic vessel. Scale bar = 500 µM. (D) Representative tail vessel pictures of Tg(l-fabp:DBP-eGFP/flk-mcherry) zebrafish with normal intersegmental tail vessel sprouting (left) and quantification of intersegmental vessels sprouting in zebrafish that were injected with miR mimics and morpholinos as indicated (right). n = 12 per group, total n = 96.

Vegf-Aa knockdown by miR-26a-5p causes proteinuria and edema in afore healthy zebrafish (A): Pictures of tail vessels (a), glomeruli fusing at zebrafish midline (b) and eye vessel plexus (c) of 48 h old zebrafish shows proper vascular development as well as glomerular fusion. Tail and eye vessel pictures were taken of a Tg(l-fabp:DBP-eGFP/Flk-mcherry) transgenic zebrafish with red fluorescent endothelium. Glomerular fusion is shown in a Tg(wt1b:eGFP) transgenic zebrafish that expresses eGFP under control of the wt1b promotor, arrow points at the fused glomerulus). Scale bar = 100 µM and 300 µM as indicated. (B,C) Quantification of loss of plasma proteins by measuring maximum eye fluorescence of the retinal vessel plexus (B) and quantification of edemous phenotype (C) of Tg(l-fabp:DBP:eGFP) zebrafish larvae at 120 hpf. A control miR mimic (miR-CTRL), a miR-26a-5p mimic (miR-26a-5p) or the combination of miR-26a-5p and Vegf-Aa protein were injected in the cardinal vein at 48 hpf. At this time the tail vasculature, glomerular fusion and retinal vessel formation was already formed; c.v. miR-CTRL n = 10, c.v. miR-26a-5p n = 17, miR-26a-5p + Vegf-Aa n = 13, WT + Vegf-Aa n = 8, total n = 48.

Vegf-Aa knockdown by vegf-Aa-MO or miR-26a-5p mimic causes glomerular endotheliosis and podocyte effacement that can be rescued by Vegf-Aa protein. Transmission electron microscopy pictures showing glomerular endotheliosis and podocyte effacement vegf-Aa knockdown by injection of a vegf-Aa morpholino (vegf-Aa-MO) or a miR-26a-5p mimic (miR-26a-5p). These pathologies were ameliorated when a zebrafish Vegf-Aa protein was co-injected. Pictures of the glomerulus were taken of 120 h old fish. Zebrafish were injected with a control morpholino (CTRL-MO), a vegf-Aa morpholino (vegf-Aa-MO), a recombinant zebrafish Vegf-Aa protein (Vegf-Aa protein), a miR-26a-5p mimic (miR-26a-5p), a control miR mimic (miR-CTRL) or a combination of vegf-Aa-MO and recombinant Vegf-Aa protein (vegf-Aa-MO + Vegf-Aa) a.e a combination of miR-26a mimic and zebrafish Vegf-Aa protein (miR-26a-5p + Vegf-Aa) as indicated at one to four cell stages or at 48 hpf (c.v. miR-26a, c.v. miR-26a-5p + Vegf-Aa). Scale bar = 500 nm.