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Osteogenesis is a highly complex and ordered developmental process in vertebrates. As a key role on osteogenesis, cbfa1 regulates its downstream genes by binding to the target DNA or forming a ternary complex in which pRb binds p204 to cbfa1. p204 is also identified as an important regulator in modulating cell proliferation, cell cycling, skeletogenesis and so on. Exogenous p204 and cbfa1 were introduced into zebrafish embryos to study their effects on skeleton development in vivo. The results showed that p204 and cbfa1 were expressed throughout the whole embryo at early embryogenesis stages, then their expression decreased gradually and focused on ventral cells and retina capsules. Malformations and differentiation of cartilage were induced by foreign p204 and cbfa1 injection, and the top level of malformation rate was induced by p204&cbfa1 double injection. Moreover, the expression of three genes (osx, tcf7 and vent) involved in osteogenesis and dorsoventral patterning were significantly upregulated after injection. Similar with the malformation rate, the top expression level of the three genes is also caused by double injection. The results of acid-free double staining showed that endochondral ossification was more obviously affected by p204&cbfa1 injection compared with other injection groups. This study showed that p204 and cbfa1 have the synergistic effect on osteogenesis and dorsoventral patterning, not only in vitro, but also in zebrafish embryogenesis.