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

新型 BMP-2 诱导基因 Wdr5 在肢体发育中的作用

• 批准号: 7137964
• 负责人: FRANCESCA GORI
• 金额: $ 17.5万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7137964
• 关键词: Retroviridae; biological signal transduction; bone morphogenetic proteins; cell differentiation; cell growth regulation; chick embryo; chondrocytes; developmental genetics; gene induction /repression; gene interaction; genetic regulation; guanine nucleotide binding protein; histogenesis; histology; in situ hybridization; limbs; nonmammalian vertebrate embryology; osteoblasts; skeletal system; transfection /expression vector; Retroviridae; biological signal transduction; bone morphogenetic proteins; cell differentiation; cell growth regulation; chick embryo; chondrocytes; developmental genetics; gene induction /repression; gene interaction; genetic regulation; guanine nucleotide binding protein; histogenesis; histology; in situ hybridization; limbs; nonmammalian vertebrate embryology; osteoblasts; skeletal system; transfection /expression vector

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.

描述（由申请人提供）：本申请研究了使用鸟类复制胜任逆转录病毒（RCAS）系统WDR5（一种新型BMP-2诱导的WD-40蛋白）WDR5的作用。 WDR5编码属于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质。该蛋白质家族已被证明在许多细胞功能中起作用，包括信号转导，mRNA加工，基因调控，囊泡运输和细胞周期调节。 WDR5在体外和体内在软骨细胞和成骨细胞中表达，并在体外加速其分化。初步研究表明，靶向WDR5表达成骨细胞会加速成骨细胞分化。这些研究中的一个新发现是肥厚的软骨细胞层的扩展，这表明骨项圈中的WDR5表达以旁分泌的方式作用以调节软骨细胞分化。尽管这些研究为WDR5在骨骼发育中的作用提供了见解，但他们尚未阐明WDR5是否对此过程至关重要。为了解决WDR5在骨骼发育至关重要的假设，将使用RCAS系统在人U6启动子的控制下表达短发夹RNA（SHRNA），以特别使整个发育中的鸡肉肢体的WDR5表达均匀。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的小鼠大，并且它在母亲之外发育的事实使研究单个基因对胚胎发育的沉默的影响变得更加容易。为了检查WDR5在体内高点rcan-shwdr5中的作用，并将含有扰乱的（c）shRNA序列（RCAN-CORTOR）逆转录病毒的RCAN注入E3.5雏鸡胚胎的右翼，通过在蛋壳中形成窗户。对翼翼将作为控制。在最初的研究中，将在E7，E8和E10上收获胚胎。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。将在组织学上研究不同程度的WDR5沉默程度的影响，并确认我们的组织学发现，将进行原位杂交。这些研究应对WDR5在胚胎发育过程中的功能进行快速有效的分析。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，并为有条件敲除小鼠的产生提供理由。