This application investigates the role of Wdr5, a novel BMP-2-induced WD-40 protein, in limb development using an avian replication competent retrovirus (RCAS) system. Wdr5 encodes for a protein belonging to the highly conserved family of WD-40 repeat proteins found in all eukaryotes. This family of proteins has been shown to play a role in numerous cellular functions including signal transduction, mRNA processing, gene regulation, vesicular trafficking and regulation of the cell cycle. Wdr5 is expressed in chondrocytes and osteoblasts in vitro and in vivo and accelerates their differentiation in vitro. Preliminary studies demonstrated that targeted expression of Wdr5 to osteoblasts accelerates osteoblast differentiation. A novel finding in these studies is the expansion of the hypertrophic chondrocyte layer, suggesting that Wdr5 expression in the bone collar acts in a paracrine fashion to regulate chondrocyte differentiation. Although these studies have provided insights into the role of Wdr5 in skeletal development they do not elucidate whether Wdr5 is essential for this process. To address the hypothesis that Wdr5 is essential in skeletal development, the RCAS system will be used to express short hairpin RNA (shRNA) under the control of the human U6 promoter to specifically silence the expression of Wdr5 throughout the developing chicken limb. Relative to the mouse system, the chicken system has advantages, including the fact that the developing chicken embryo is larger than the developing mouse, and the fact that it develops outside the mother makes it much easier to investigate the effects of the silencing of individual genes on embryonic development. To examine the role of Wdr5 on limb development in vivo high-titer RCAN-shWdr5 and RCAN containing a scrambled (c) shRNA sequence (RCAN-control) retroviral inoculates will be injected into the right wing of E3.5 chick embryos by creating a window in the eggshell. Contralater wings will serve as control. In initial studies, embryos will be harvested at E7, E8 and E10. Based on the results of these initial studies, analyses will be performed at earlier time points and/or more frequent intervals. The effects of the differing degrees of Wdr5 silencing will be investigated histologically and to confirm our histological findings, in situ hybridization will be performed. These studies should offer a rapid and efficient analysis of the function of Wdr5 during embryonic development. Furthermore, they will allow us to dissect the roles of other genes that interact with Wdr5 and will provide a rationale for the generation of conditional knockout mice. ? ? ?
| Zhu, Shimei; Zhu, Eric D; Provot, Sylvain et al. (2010) Wdr5 is required for chick skeletal development. J Bone Miner Res 25:2504-14 |
