MSX2 AND DLX-5 IN LIMB PATTERN FORMATION AND CARTILAGE DIFFERENTIATION

项目1的目标是研究角色和角色之间的关系 同源框基因MSX2和D1X-5在花纹形成调控中的作用 雏鸡肢芽发育过程中的软骨分化。MSX2和 D1X-5是MSH和Distal-less家族的成员，在 肢芽的顶端外胚层脊(AER)，起着至关重要的作用 在引导LIB的生长和模式方面，也是共同表达的 在肢芽的前中胚层，在正常发育期间 几乎不会生长，也不会形成骨骼结构， 这表明这些基因可能密切参与调控 控制AER活动和指定前部非骨骼的网络 唇芽中胚层形成区。为了研究这些可能性，MSX2和 D1X-5逆转录病毒表达载体将用于检测 这些基因的局部异位表达，单独和组合，在 小鸡翅芽的远端前外胚层彼此相连 表达；关于自己的表达；关于基因的表达 信号分子，如成纤维细胞生长因子-4、成纤维细胞生长因子-2或WNT-5a 与调节AER活性有关；以及，关于形态发生和基因 在下面的前中胚层中有表达。本地化的影响 MSX2和D1X-5的异位表达，单独和组合在 肢芽后中胚层对形态发生和基因表达的影响 也将进行研究。除了在AER和 肢芽的前中胚层，D1X-5在 软骨化开始时肢芽的近端中央核心 肢体骨骼元素的分化及其启动 几种软骨特异性基质蛋白的基因表达。 D1X-5的表达与肿瘤的发病也有很强的相关性 软骨特异性基因的体外表达。DIX-5表达载体 将被用来确定D1x-5是否可以促进 软骨特异的基质蛋白和可调控表达的报告 含有II型启动子和增强子元件的构建体 胶原蛋白基因。如果是这样的话，D1X-5蛋白与推测的 第一个内含子增强子元件中的同源结构域结合序列 II型胶原基因的体外和体内研究将进行。取决于 根据前面实验的结果，将启动研究以 寻找D1X-5在肢体分化中的其他潜在靶基因 D1X-5可能与软骨和蛋白质相互作用调控基因 在这些细胞中表达。

The goal of Project 1 is to study the roles and relationships between the homeobox-containing genes Msx2 and D1x-5 in regulation of pattern formation and cartilage differentiation in the developing chick limb bud. msx2 and D1x-5, members of the Msh and Distal-less families, are co-expressed in the apical ectodermal ridge (AER) of the limb bud, which plays a crucial role in directing outgrowth and patterning of the lib, and are also co-expressed in the anterior mesoderm of the limb bud, which during normal development undergoes little outgrowth and fails to give rise to skeletal structures, suggesting that these genes may be intimately involved in regulatory networks controlling AER activity and specifying the anterior non-skeletal forming region of lib bud mesoderm. To study these possibilities, Msx2 and D1x-5 retroviral expression vectors will be used to examine the effect of localized ectopic expression of these genes, alone and in combination, in the distal anterior ectoderm of the chick wing bud on one another's expression; on their own expression; on the expression of genes for signalling molecules, such as FGF-4, FGF-2, or Wnt-5a that have been implicated in mediating AER activity; and, on morphogenesis and gene expression in the underlying anterior mesoderm. Effects of localized ectopic expression of Msx2 and D1x-5, alone and in combination, in the posterior mesoderm of the limb bud on morphogenesis and gene expression will also be studied. In addition to being expressed in the AER and anterior mesoderm of the limb bud, D1x-5 is expressed at high amounts in the proximal central core of the limb bud at the onset of the chondrogenic differentiation of the skeletal elements of the limb and the initiation of the expression of genes for several cartilage-specific matrix proteins. There is also a strong correlation between D1x-5 expression and the onset of cartilage-specific gene expression in vitro. Dix-5 expression vectors will be used to determine if D1x-5 can promote the expression of genes for cartilage-specific matrix proteins and can regulate expression of reporter constructs containing promoter and enhancer elements of the type II collagen gene. If so, the interaction of D1x-5 protein with putative homeodomain binding sequences in the first intron enhancer element in the type II collagen gene in vitro ad in vivo will be studied. Depending on the outcome of the preceding experiments, studies will be initiated to identify other potential target genes of D1x-5 in differentiating limb cartilage and proteins with which D1x-5 might interact to regulate gene expression in these cells.