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.
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