Role of Wdr5, a Novel BMP-2 Induced Gene, in Limb Development

描述（由申请人提供）：本申请使用禽类复制型逆转录病毒（RCAS）系统研究了Wdr 5（一种新型BMP-2诱导的WD-40蛋白）在肢体发育中的作用。Wdr 5编码属于在所有真核生物中发现的高度保守的WD-40重复蛋白家族的蛋白。该蛋白质家族已被证明在许多细胞功能中发挥作用，包括信号转导、mRNA加工、基因调控、囊泡运输和细胞周期调控。Wdr 5在体外和体内的软骨细胞和成骨细胞中表达，并在体外加速它们的分化。初步研究表明，Wdr 5的靶向表达成骨细胞加速成骨细胞的分化。这些研究中的一个新发现是肥大软骨细胞层的扩张，表明骨领中的Wdr 5表达以旁分泌方式调节软骨细胞分化。虽然这些研究提供了深入了解Wdr 5在骨骼发育中的作用，但它们并没有阐明Wdr 5是否对这一过程至关重要。为了解决Wdr 5在骨骼发育中必不可少的假设，RCAS系统将用于在人U6启动子的控制下表达短发夹RNA（shRNA），以在整个发育的鸡肢中特异性沉默Wdr 5的表达。相对于小鼠系统，鸡系统具有优势，包括发育中的鸡胚胎比发育中的小鼠大，并且它在母体外发育的事实使得研究单个基因沉默对胚胎发育的影响变得更加容易。为了检查Wdr 5在体内肢体发育中的作用，将高滴度RCAN-shWdr 5和含有乱序（c）shRNA序列的RCAN（RCAN-对照）逆转录病毒接种物通过在蛋壳中创建窗口注射到E3.5鸡胚的右翼中。双翅将作为控制。在最初的研究中，胚胎将在E7，E8和E10收获。根据这些初步研究的结果，将在更早的时间点和/或更频繁的时间间隔进行分析。Wdr 5沉默的不同程度的影响将进行组织学研究，并确认我们的组织学结果，原位杂交将进行。这些研究将为Wdr 5在胚胎发育过程中的功能提供快速有效的分析。此外，它们将使我们能够剖析与Wdr 5相互作用的其他基因的作用，并为条件性敲除小鼠的产生提供理论基础。

DESCRIPTION (provided by applicant): 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.