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

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）：本申请利用禽类复制能力逆转录病毒（RCAS）系统研究了Wdr5（一种新型BMP-2诱导的WD-40蛋白）在肢体发育中的作用。 Wdr5 编码的蛋白质属于所有真核生物中高度保守的 WD-40 重复蛋白家族。该蛋白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。 Wdr5 在体外和体内的软骨细胞和成骨细胞中表达，并加速它们的体外分化。初步研究表明，Wdr5向成骨细胞的靶向表达可加速成骨细胞分化。这些研究的一个新发现是肥大软骨细胞层的扩张，表明骨领中 Wdr5 的表达以旁分泌方式调节软骨细胞分化。尽管这些研究深入了解了 Wdr5 在骨骼发育中的作用，但它们并未阐明 Wdr5 是否对该过程至关重要。为了解决 Wdr5 在骨骼发育中至关重要的假设，RCAS 系统将用于在人 U6 启动子的控制下表达短发夹 RNA (shRNA)，以特异性沉默整个发育中鸡肢的 Wdr5 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更大，而且它在母体外发育，这使得研究个体基因沉默对胚胎发育的影响变得更加容易。为了检查 Wdr5 对体内肢体发育的作用，通过在蛋壳中创建一个窗口，将高滴度 RCAN-shWdr5 和含有乱序 (c) shRNA 序列的 RCAN（RCAN 对照）逆转录病毒接种物注射到 E3.5 鸡胚的右翼中。对侧机翼将作为控制装置。在初步研究中，胚胎将在 E7、E8 和 E10 时收获。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。将从组织学角度研究不同程度的 Wdr5 沉默的影响，并进行原位杂交以证实我们的组织学发现。这些研究应该能够快速有效地分析 Wdr5 在胚胎发育过程中的功能。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，并为生成条件性基因敲除小鼠提供理论依据。