Characterization of regulatory elements in the medaka osterix promoter required for osteoblast expression

The zinc finger transcription factor Osterix/Sp7 is an essential regulator of osteoblastogenesis. In mammals, osterix expression is regulated by Runx2, Msx2 and Dlx5 but recent findings suggest that also retinoic acid plays an important role for osteoblast differentiation and function. Yet, how these and other factors act on the osterix promoter is largely unknown. Expression, knock-down and promoter analyses have indicated that the function of Osterix in osteoblasts is conserved in teleosts and mammals. In the present study, we have used the teleost medaka to identify and characterize a region containing potential retinoic acid response elements in the osterix promoter. We analysed whether this region is important for activity in osteoblasts in vivo, using transgenic medaka lines with modified osterix promoter regions. Promoter activity in vivo and in vitro revealed a short nucleotide sequence in the promoter with crucial positive regulatory function. Mutations of this element lead to a complete inactivation of the osterix promoter in osteoblasts and made it insensitive to retinoic acid treatment. The comparison with the regulatory regions of osterix in other species suggests that the function of this element is highly conserved in vertebrates.