| There are three specific cell types in skeleton:chondrocyte in cartilage, osteoblast and osteoclast in bone. Genetic and molecular analysis of bone morphogenetic proteins (BMPs) has indicated that they are vital regulators in the process of skeletal patterning by controlling the formation and shape of various skeletal mesenchymal condensations. cbfa1 is identified as an important factor in chondrocyte and osteoblast differentiation. As an important regulator of skeletal differentiation, p204 modulates a series of biochemical mechanisms to promote the differentiation of different tissues. It is found that cbfa1 homologous gene(runx2b) exists in zebrafish genome, but the p204 orthologs cannot be found by genetic comparison and analysis between zebrafish and mice.Although zebrafish has not been used widely for study on skeletal development and disease. Zebrafish is a necessary and valuable vertebrate complement to the mouse model, and several studies have showed its potential. Moreover, its cranial neural crest plays a similar role as in tetrapods indicating that most of the skull is neural crest derived.To study the role of p204 cooperated with cbfa1 on osteogenesis and embryogenesis in vivo, exogenous p204-EGFP and cbfa1-EGFP were introduced into one cell stage embryos of zebrafish. The GFP expression were observed by fluorescence microscope. We selected 4 genes (osx, vent, runx2b and tcf7), the key regulators in zebrafish osteogenesis at different development stages, as taget genes to assay their expression levels involved in zebrafish dorsoventral patterning and osteoblastogenesis using qPCR. To further investigate expression levels of osx and tcf7, whole-mount in situ hybridization was performed. Besides, the acid-free double staining was carried out to examine zebrafish osteogenesis.The results showed that various malformations were induced by p204 and cbfa1 injection, including pericardial sac-expansion, cyrtosis and short/bent tails. 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 as malformation rate, the top expression level of the three genes was also caused by double injection. Meanwhile, exogenous p204 and cbfal injection could induce the differentiation of cartilage(especially pharyngeal arches) and stimulate bone formation. This study showed that p204 and cbfal have some synergistic effect on osteogenesis and dorsoventral patterning, not only in vitro, but also in zebrafish embryogenesis. |
