MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

这个项目的主要目标是研究分子调控。 同源异型盒相关基因MSX-2(和D1X-5和/或MSX-1)在 发育中的脊椎动物肢体。MSX-2和MSX-1是MSH-1的成员 就像基因家族一样，都与肢体的调节有关 形态发生和图案形成。MSX-2和MSX-1是相互作用的 由顶端外胚层脊(AER)和下面的亚脊表达 中胚层细胞发育的肢芽，以及各种研究都有 提示这些基因参与了相互作用。 AER和肢体中胚层之间的关系，这是肢体生长所必需的 图案化。此外，MSX-2和MSX-1在前角共同表达。 肢芽的中胚层，在那里它们可能参与了 四肢中胚层的前部非骨骼形成区 发生程序性细胞死亡的肢芽区域，即 参与塑造肢体的轮廓。D1X-5在ALL中共表达 MSX-2结构域，也表达在凝集性间充质中 四肢正在经历软骨生成。Tahe基因突变表达的研究 突变型肢芽中MSX-2的模式显示，AER和 其表达的中胚层结构域是独立调节的。这个 该项目的主要初始目标是确定监管机构 MSX-2基因负责其空间特定结构域的元件 以及它对其他相关调节分子的反应 在肢体图案上。为此，基因组克隆包含完整的 鸡MSX-2基因加上15kb的5‘侧翼序列和11kb的3’端序列 已分离出侧翼序列，并研究了MSX-2的调控。 2转基因小鼠的基因表达已启动。这些研究 已经确定了其AER所需的MSX-2基因的350bp区域 转基因小鼠的表达结构域和附加的顺式作用区域 哪些定义MSX-2基因表达的调节区将是 已确认身份。核酸调节因子相互作用的部位 空间上特定的顺式作用区域将被识别和表征。 MSX-2基因中的调控元件也将通过DNA酶进行鉴定 不同肢体核提取液的变态反应分析 地区。类似的方法将被用来描述监管机构 D1x-5和/或MSX-1基因中的元件。这些研究应该提供 深入了解MSX-2(以及D1X-5和MSX-1)基因是如何调控的，以及 监管网络如何参与模式的形成 发育中的肢体。

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.