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Morpholino knockdown of twist1a and twist1b induces the appearance of ventralized embryos and increases the expression of runx2b in zebrafish. A, Antisense morpholinos (MO) for twist1a (twist1a atgMO), twist1b (twist1b atgMO), twist2 (twist2 atgMO) and twist3 (twist3 atgMO), were designed against the 52 UTR and ATG regions, which blocked translation of each transcript. Each atgMO was microinjected into 1-cell to 4-cell embryo and the percentage of each dorsoventral patterning was calculated at 36–48 hpf. B, Representative pictures of induced Class 1–5 ventralized (V1-5/no defect) and dorsalized (D) embryos. V and D phenotype annotations were described in ref. 20 and 21, respectively. C, Table summarizing the ventralized or dorsalized embryo features of zebrafish microinjected with indicated concentration of each atgMO with or without twist1a/1b mRNA or T2 runx2b atgMO. Zebrafish were microinjected with MO-SC, (D) twist1a atgMO or (E) twist1b atgMO with or without (D) twist 1a mRNA or (E) twist 1b mRNA and quantitative RT-PCR for T1 runx2b and T2 runx2b were 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; # p<0.05 and ## p<0.01 versus twist1a/1b atgMO).
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