Evaluation of candidate miR function during zebrafish development. (A) MiR kinetics were evaluated during early zebrafish development. Total RNA was isolated on respective timepoints (hours post fertilization; hpf) for qRT-PCR. (B) MiR-20b, -30a, -30b, and -128a were upregulated in developing zebrafish larvae from 24 hpf increasing until 72 hpf. (C) Morpholino oligos (MOs) were injected in 1–2 cell-stage zebrafish embryos and miR knockdown, morphological changes, fractional shortening, and beating rate was analyzed (24, 48, 72 hpf). (D) MiR expression (evaluated by qRT-PCR) in zebrafish larvae was sufficiently reduced by MOs at 24 hpf by more than 85% for all candidate miRs in comparison to controls without MOs (t-test). (E) Morphological changes of MO-morphants at 48 hpf. MO-20b morphants showed cerebral hemorrhage (red arrow) and edema (edema in the eye region indicated by a white arrow). MO-30b morphants developed malformations and edemas (red arrow highlights a visible edema and enlarged hydrocephalus). MO-128a larvae exhibited a robust pericardial edema (red arrow) and blood congestion at the right outflow tract of the heart (white arrow). Scale bars: 250 µm. (F) Cardiac phenotype of MO-128a treated Tg(myl7:ras-GFP) zebrafish larvae at 48 hpf including smaller ventricles (white arrow) and abnormalities in heart looping. Scale bars: 5 µm. (G) MO-128a larvae (n = 8) showed a significantly reduced fractional shortening at 72 hpf compared to non-treated larvae (w/o MO, n = 10) (t-test) (H) MO-128a morphants (n = 11) appeared to have a significantly slower heart rate at 72 hpf compared to the controls (w/o MO, n = 11) (t-test). All data are represented as means ± SEM. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
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