In the current study, we first found that in zebrafish under hypoxic conditions, bone mineralization was inhibited and T1 and T2 Diatrizoic acid runx2b and their downstream targets were downregulated, while hif-1a and twist were upregulated. Inhibition of twist1a and twist1b by morpholino oligonucleotides increased the expression of T1 and T2 runx2b, and induced ventralized patterning, while microinjecting zebrafish embryos with full length twist1a and twist1b mRNA decreased the expression of T1 and T2 runx2b, and induced dorsalized patterning in zebrafish. Twist1a and twist1b morphlinos also rescued hypoxiainduced decrease in craniofacial skeletal development in zebrafish. Twist is known to trigger epithelial-mesenchymal transition mechanisms and increase cells with migratory ability. Although Twist is constantly expressed in various cells including osteoblasts, its roles in skeleton development is seldom, if ever, investigated. Previous studies have shown that TWIST silencing enhanced osteoblast gene expression and matrix mineralization. In this current study, we demonstrate that twist plays an essential role in skeleton development and axis establishment by regulating runx2b in zebrafish. Interestingly, only TWIST in human MSCs and twist1a and twist1b, but not twist2 and twist3, in zebrafish possess these functions. Moreover, RUNX2 in human and runx2b in zebrafish are mainly involved in skeleton development or axis establishment, suggesting the conservation of Twist-Runx2 pathway in mammalian cells and zebrafish. Previously, Twist has been demonstrated to inhibit DNA binding and gene activation by Runx2, while Runx2 expression was not affected in mice carrying Twist heterozygosity. The current study found Nortriptyline overexpression and knockdown of Twist increased and decreased the expression of Runx2 both at the mRNA and protein levels, respectively. The discrepancy between our study and the previous one may be because the suppression of Twist expression by Twist heterozygosity in mice is not sufficient to downregulate Runx2, as is the minimal effect of the low dose of twist1b atgMO to increase runx2b expression. Twist has been reported to have a synergistic effect with dorsal and snail in integrating diverse dorsoventral patterning in the Drosophila embryo.