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Morpholino knockdown of twist1a and twist1b induces runx2b expression and promotes bone formation in zebrafish. (A to D) Zebrafish were microinjected without (No injection, NI) or with control MO (MO-SC), twist1a or twist1b atgMOs and runx2b expression was analyzed by in situ hybridization at 8 hpf (A), 14 hpf (B), and 48 hpf (C) with runx2b probe. Pictures of lateral (L), animal pole (AP), and head region dorsal (HD) views are displayed. Runx2b expression was induced by twist1a or twist1b atgMO compared to MO-SC at 8 hpf, 14 hpf and 48 hpf. (arrow, ventral region; arrowhead, dorsal region; fb, forebrain area; hb, hindbrain area; ceratobranchial 1–5, Cb1-5; cleithrum, Cl) D, Quantitative RT-PCR for osterix and col10a1 was performed at 8, 14 and 48 hpf (n = 3). Results are shown as the relative expression to β-actin (mean ± SD) and significance was determined by Student′s t-test. (* p<0.05 and ** p<0.01 versus MO-SC). E, F, Zebrafish survived after microinjection with MO-SC or twist1a or twist1b atgMO were cultured with or without 100 μM DFX and bone mineralization was analyzed by (E) ARS staining and (F) calcein labeling at 8 dpf. The number of positive staining developing centra form ring with ARS or calcein labeling were calculated.
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