MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

该项目的主要目标是研究分子调控 同源框相关基因Msx-2（和D1 x-5和/或Msx-1）在 发育中的脊椎动物肢体 Msx-2和msx-1是msh- 就像基因家族一样，与肢体的调节有关， 形态发生和图案形成。 Msx-2和msx-1是 由顶端外胚层嵴（AER）和下面的亚嵴表达 中胚层细胞的发展肢芽，和各种研究， 表明这些基因参与了相互作用， 在AER和肢体中胚层之间，这是肢体生长所必需的， 模式化 此外，Msx-2和Msx-1在前额叶共表达。 肢芽的中胚层，在那里他们可能参与的规格 肢中胚层的前非骨形成区， 肢芽中发生程序性细胞死亡的区域， 参与塑造肢体的轮廓。 D1 x-5在所有细胞中共表达， Msx-2结构域，也表达于血管的致密间充质中。 四肢正在进行软骨形成。 tahe基因表达改变的研究 Msx-2在突变肢芽中的模式揭示了AER和 其表达的中胚层结构域是独立调节的。 的 该项目的主要初始目标是确定监管 Msx-2基因的元件，负责其空间特异性结构域 表达和对其他相关调节分子的反应 在肢体模式中。 为此，含有完整的 鸡Msx-2基因加上15 kb的5'侧翼序列和11 kb的3'侧翼序列， 侧翼序列已经被分离出来，并对Msx- 2基因在转基因小鼠中的表达已经启动。 这些研究 已经鉴定了其AER所需的msx-2基因的350 bp区域 在转基因小鼠中的表达结构域，以及另外的顺式作用区 其定义Msx-2基因表达的调节区域， 鉴定 核酸调节因子相互作用的位点 将鉴定和表征空间特异性顺式作用区。 Msx-2基因内的调控元件也将通过DNase鉴定 使用来自不同肢体的核提取物的超敏反应分析 地区 类似的方法将用于描述监管 D1 x-5和/或Msx-1基因中的元件。 这些研究将提供 深入了解Msx-2（以及D1 x-5和Msx-1）基因是如何调控的， 监管网络如何参与模式的形成 发展肢体。

The broad goal of this project is to investigate the molecular regulation of the homeobox-related genes Msx-2 (and D1x-5 and/or Msx-1) in the developing vertebrate limb. Msx-2 and msx-1, which are member of the msh- like family of genes, have been implicated in the regulation of limb morphogenesis and pattern formation. Msx-2 and msx-1 are reciprocally expressed by the apical ectodermal ridge (AER) and the underlying subridge mesodermal cells of the developing limb bud, and a variety of studies have suggested that these genes are involved in the reciprocal interactions between the AER and limb mesoderm that are required for limb outgrowth and patterning. In addition, Msx-2 and Msx-1 are co-expressed in the anterior mesoderm of the limb bud, where they may be involved in specification of the anterior non=-skeletal-forming region of limb mesoderm, and in discrete regions of the limb bud in which programmed cell death is occurring that is involved in shaping the contours of the limb. D1x-5 is co-expressed in all Msx-2 domains and is also expressed in the condensing mesenchyme of the limb undergoing chondrogenesis. Studies on tahe altered expression patterns of Msx-2 in mutant limb buds have revealed that the AER and mesodermal domains of its expression are independently regulated. The primary initial goal of this project is to identify the regulatory elements of the Msx-2 gene responsible for its spatially specific domains of expression and for its response to other regulatory molecules involved in limb patterning. To this end, genomic clones containing the full chicken Msx-2 gene plus 15 kb of 5' flanking sequence and 11 kb of 3' flanking sequence have been isolated, and studies on the regulation of Msx- 2 gene expression in transgenic mice have been initiated. These studies have identified a 350 bp region of the msx-2 gene required for its AER domain of expression in transgenic mice, and additional cis-acting regions which define the regulatory regions of the Msx-2 gene expression will be identified. Sites of nucleic acid regulatory factor interactions within spatially-specific cis-acting regions will be identified and characterized. Regulatory elements within the Msx-2 gene will also be identified by DNase hypersensitivity analyses using nuclear extracts from different limb regions. Similar approaches will be used to characterize regulatory elements in the D1x-5 and/or Msx-1 genes. These studies should provide insight into how the Msx-2 (and D1x-5 and Msx-1) genes are regulated and how the regulatory network is involved in pattern formation in the developing limb.