MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

项目3(William B.Bording，PI)的主要目标将是 研究MSX2基因表达的转录调控。 发展脊椎动物肢体，特别强调识别 顺式调控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结构域 AER和肢体中胚层。已经有两个独立的增强剂元素 在MSX2基因中发现将报告基因的表达定向到 转基因小鼠的AER。转基因小鼠中的报告构建将 用来进一步定义这两个AER增强子元素并检查 这两个站点之间的功能相互作用。类似的方法 将被用来研究DNA酶序列 目前在MSX2基因中发现的过敏性位点 支撑期在调节空间特异度方面发挥作用 MSX2表达的结构域。1-杂交筛选过程或 将使用西南筛选技术来确定候选人 与增强子区域相互作用的反式作用分子 在MSX2基因中被鉴定。其中涉及的调控序列 MSX2基因对Dlx5和Dlx5等其他调控基因的反应 信号，如Sonic Hedgehog、BMP-4、IGF-1和FGFs 参与调节AER活动和/或肢体模式的也将是 通过确定这些因素对 多种MSX2报告基因在培养细胞中的表达 四肢细胞。此外，MSX2在基因表达调控中的作用 在BMP-1中，可能是肢体中MSX2的下游靶点，将是 MSX2表达载体与MSX2表达载体共转染实验研究 Bmp4启动子报告基因构建。这些研究应该提供 深入了解MSX基因是如何调控的，以及如何调控 网络参与了肢体发育过程中图案的形成。

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.