MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

项目3的主要目标（威廉B。支持，PI）将是 研究Msx 2表达的转录调控， 发展脊椎动物肢体，特别强调识别 顺式调节元件和反式作用因子， Msx 2表达的独立调节的空间特异性结构域， AER和四肢中胚层。两个单独的增强子元件已经被 在Msx 2基因中鉴定出指导报告基因表达， 转基因小鼠的AER。转基因小鼠中的报告基因构建体将 用于进一步定义这两个AER增强子元件并检查 两个位点之间的功能性相互作用。类似的方法 将被用来研究DNA酶的序列 在目前的Msx 2基因中发现的超敏位点 支持期发挥调节作用的空间特异性 Msx 2的表达。1-杂交筛选过程或 西南筛选技术将用于确定候选人 与增强子区域相互作用的反式作用分子 Msx 2基因中的一种。参与的调控序列 Msx 2基因对其他调节基因如Dlx 5和 信号传导，如Sonic hedgehog，BMP-4，IGF-1和FGF， 参与调节AER活性和/或肢体模式的基因也将被 通过确定这些因素对 多种Msx 2报告基因构建体在培养的细胞中的表达 肢体细胞此外，Msx 2在调节表达中的作用 BMP-1是肢体中msx 2的假定下游靶点， 在使用Msx 2表达载体的共转染实验中研究， BMP 4启动子报告基因构建体。这些研究将提供 深入了解Msx基因是如何调控的，以及调控基因是如何表达的。 网络参与了发育肢体的图案形成。

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.