MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

同源盒基因参与肢体模式形成的分子机制

• 批准号: 6311633
• 负责人: WILLIAM B UPHOLT
• 金额: $ 13.33万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311633
• 关键词: DNA binding protein; bone morphogenetic proteins; chick embryo; developmental genetics; ectoderm; epidermal growth factor; fibroblast growth factor; gene expression; genetic enhancer element; genetic library; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; homeobox genes; insulinlike growth factor; laboratory mouse; limbs; mesoderm; molecular genetics; reporter genes; tissue /cell culture; transcription factor; transfection; vertebrate embryology

The major goal of Project 3 (William B. Uphold, PI) will be to investigate the transcriptional regulation of Msx2 expression in the developing vertebrate limb with particular emphasis on identification of cis-regulatory elements and trans-acting factors that control the independently-regulated spatially-specific domains of Msx2 expression in the AER and limb mesoderm. Two separate enhancer elements have been identified in the Msx2 gene that direct expression of reporter genes to the AER of transgenic mice. Reporter constructs in transgenic mice will be used to define further these two AER enhancer elements and to examine the functional interactions between the two sites. Similar approaches will be used to study the possibility that sequences at DNase hypersensitive sites identified in the Msx2 gene during the current period of support play a role in regulating the spatially-specific domains of Msx2 expression. The 1-hybrid screening process or the Southwestern screening technique will be used to identify candidate trans-acting molecules that interact with the enhancer regions identified in the Msx2 gene. The regulatory sequences involved in the response of the Msx2 gene to other regulatory genes such as Dlx5 and signaling such as Sonic hedgehog, BMP-4, IGF-1, and FGFs that are involved in regulating AER activity and/or limb patterning will also be investigated by determining the effect of these factors on the expression of a variety of Msx2 reporter gene constructs in cultured limb cells. In addition, the role of Msx2 in regulating the expression of the BMP-1, a putative downstream target of msx2 in the limb, will be studied in co-transfection experiments using Msx2 expression vectors and BMP4 promotor reporter gene constructs. These studies should provide insight into how the Msx gene is regulated and how the regulatory network is involved in pattern formation in the developing limb.

项目 3（William B. Uphold，PI）的主要目标是 研究Msx2表达的转录调控 发育脊椎动物肢体，特别强调识别 顺式调节元件和反式作用因子控制 Msx2表达的独立调控的空间特异性域 AER 和肢体中胚层。两个独立的增强子元件已被 在 Msx2 基因中发现，该基因指导报告基因的表达 转基因小鼠的 AER。转基因小鼠中的报告构建体将 用于进一步定义这两个 AER 增强子元件并检查 两个站点之间的功能交互。类似的方法 将用于研究 DNase 序列的可能性 在当前的研究中，Msx2 基因中发现了过敏位点 支持期在调节空间特定性方面发挥作用 Msx2 表达域。 1-杂交筛选过程或 西南筛选技术将用于识别候选人 与增强子区域相互作用的反式作用分子 在 Msx2 基因中鉴定。涉及的调控序列 Msx2 基因对其他调控基因（例如 Dlx5 和 信号传导，例如 Sonic Hedgehog、BMP-4、IGF-1 和 FGF 参与调节 AER 活动和/或肢体模式也将 通过确定这些因素对 多种 Msx2 报告基因构建体在培养物中的表达 肢体细胞。此外，Msx2在调节表达中的作用 BMP-1（肢体中 msx2 的假定下游目标）的 使用 Msx2 表达载体进行共转染实验研究 BMP4 启动子报告基因构建体。这些研究应该提供 深入了解 Msx 基因的调控方式以及调控机制 网络参与发育中肢体的模式形成。