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

描述(由申请人提供)：本申请研究WDR5，一种新型的BMP-2诱导的WD-40蛋白，在使用禽类复制能力逆转录病毒(RCAS)系统的肢体发育中的作用。WDR5编码一种蛋白质，属于高度保守的WD-40重复蛋白家族，在所有真核生物中都存在。该家族蛋白在多种细胞功能中发挥作用，包括信号转导、信使核糖核酸的处理、基因调控、囊泡运输和细胞周期调控。WDR5在体外和体内表达于软骨细胞和成骨细胞，并在体外促进其分化。初步研究表明，WDR5靶向表达成骨细胞可加速成骨细胞分化。这些研究中的一个新发现是肥大的软骨细胞层的扩张，表明WDR5在骨领中的表达以旁分泌的方式调节软骨细胞的分化。尽管这些研究对WDR5在骨骼发育中的作用提供了洞察力，但它们并没有阐明WDR5是否对这一过程是必要的。为了解决WDR5在骨骼发育中必不可少的假设，将使用RCAS系统在人U6启动子的控制下表达短发夹状RNA(ShRNA)，以特异性沉默WDR5在整个发育鸡腿中的表达。与老鼠系统相比，鸡系统具有优势，包括发育中的鸡胚胎比发育中的老鼠大，而且它是在母亲之外发育的，这使得研究单个基因沉默对胚胎发育的影响变得容易得多。为了检测WDR5在体内肢体发育中的作用，我们将通过在蛋壳中开一个窗口，将含有打乱的(C)shRNA序列(rcan对照)的高滴度rcan-shWdr5和rcan逆转录病毒疫苗注射到E3.5鸡胚胎的右翼。对抗者的翅膀将充当控制者。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，将在较早的时间点和/或更频繁的间隔进行分析。不同程度的WDR5沉默的影响将进行组织学研究，并将进行原位杂交，以证实我们的组织学结果。这些研究应该为WDR5在胚胎发育过程中的功能提供快速而有效的分析。此外，它们将使我们能够剖析与WDR5相互作用的其他基因的作用，并将为条件基因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.