The Important Role Of Transcription Factor Sp7 / Osterix In Zebrafish Bone Development

Osteoblast differentiation is a multi-step process wherein mesenchymal cells differentiate into a series of osteocyte cell lineages. As an osteoblast-specific transcription factor, SP7/OSTERIX plays an important role in differentiation of pre-osteoblasts into mature osteoblast cells. Vertebrate SP7 is highly conserved because the amino acid sequences of the three zinc fingers are almost identical among human,mouse and zebrafish. Human osteogenesis imperfecta(OI) is resulted form various SP7 point mutations, implicating an important role of SP7 in human bone development. Due to the fact that the Sp7-/- mice died within 15 min of birth, current studies of the Sp7 functions are limited only in osteoblast cell lines or tissue-specific Sp7 conditional mice.Thus the role of Sp7 in the whole process of bone development is still unclear.The zebrafish(Danio rerio) as a model organism has been widely used in developmental and genetic studies. Here we used TALEN to generate sp7 mutant zebrafish. We observed that in contrast to Sp7 knockout mice, the homozygous sp7 mutant fish can live as long as one year. The sp7 mutant fish exhibit curved tail fin,missing opercula and the upper jaw, as shown by Alizarin red. The body weight and body length of mutant fish are significantly reduced compared with the wild types. The micro-CT analysis also found that the bone volume is significantly reduced in sp7 mutant fish. On the other hand, Alcian blue staning showed there is no significant difference in cartilage in sp7 mutant fish and wild types. These results suggest that Sp7 is essential for bone development but has no effect on cartilage development. The quantitative RT-PCR analysis showed down-regulation of the osteocalcin, spp1, phex,col1a1 a and sost fish but no altered expression of sox9 a and sox9 b in sp7 mutant.Mouse Col10a1 was known to contribute to cartilage development. Our quantitative RT-PCR and the WISH analyses showed down-regulation of col10a1 in sp7mutant, suggesting that it may play a role in zebrafish bone development. We analyzed the col10a1 promoter and found that it contains the Sp1 sites that can be bound by Sp7.Cell transfection and ChIP assays showed that Sp7 can promote the expression ofcol10a1 via binding to the Sp1 sites. Rescue assays showed that col10a1 mRNA level in sp7 mutant fish can be signifcantly restored by microinjection of normal sp7 mRNA.These results suggest that col10a1 is a new target gene of Sp7 in zebrafish.In this study, we used TALEN to generate zebrafish sp7 mutants. Characterization of sp7 mutant fish showed that Sp7 is essential for zebrafish bone develoment but not for cartilage development. We also found that col10a1 encoding X type collagen contributes to zebrafish bone development, rather than cartilage development, and is also a new target of zebrafish Sp7. Zebrafish sp7 mutant fish provide animal models for investigating novel aspects of bone development as wells as drug screens for human Osteogenesis Imperfecta(OI).