Hypoxia inhibits mineralization but not chondrogenesis in zebrafish.

A, D, G Zebrafish at 2 days post-fertilization (dpf) were treated with 5% O₂ for 1 day or B, E, H indicated concentration of DFX for 6 days and stained with (A, B) ARS, (D, E) calcein labeling, or (G, H) Alcian blue staining at 8 dpf. Mineralization was indicated by positive ARS staining or calcein labeling in the spine area (arrow) and the number of positive staining developing centra form ring with ARS or calcein labeling were calculated (C, F).

Hypoxia inhibits T1 and T2 runx2b expression in zebrafish.

Zebrafish at 48 hpf were treated with 21% O_2, 5% O₂ or indicated concentration of DFX for 1 day, and gene expression of T1 and T2 runx2b was assayed by quantitative RT-PCR. Results are shown as relative expression to ²-actin (mean ± SD) and significance was determined by Student′s t-test. (* p<0.05 and ** p<0.01 versus 21% O₂).

Hypoxia increases the expression of hif1-α and twist in zebrafish.

Zebrafish at 48 hpf were treated with or without 21% O_2, 5% O₂ or indicated concentration of DFX for 1 day and the expression of hif-1α, twist1a, twist1b, twist2 and twist3 genes was assayed by quantitative RT-PCR. Results are shown as the relative expression to ²-actin (mean ± SD) and significance was determined by Student′s t-test. (* p<0.05 and ** p<0.01 versus 21% O₂).

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).

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.

Morpholino knockdown of twist2 or twist3 induces no changes in runx2b transcription. Zebrafish were microinjected with twist2 and twist3 atgMOs and runx2b expression was analyzed by in situ hybridization at 48 hpf (n = 40 for each). Pictures of lateral (L) and dorsal views (D) are displayed.