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

项目1的目标是研究 含同源框基因Msx2和D1x-5调控模式形成 和软骨分化。 MSX2和 D1x-5是Msh和Distal-less家族的成员，在细胞中共表达。 肢芽的顶端外胚层嵴（AER），起着关键作用 在引导lib的生长和图案化中， 在肢芽的前中胚层，在正常发育期间， 几乎没有生长，不能产生骨骼结构， 这表明这些基因可能密切参与调节 控制AER活动并指定前部非骨骼肌的网络 叶芽中胚层形成区。 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查 这些基因的局部异位表达，单独和组合， 小鸡翅芽的远端前外胚层 表达;对自己的表达;对基因的表达， 信号分子，如FGF-4、FGF-2或Wnt-5a， 参与介导AER活性;以及，形态发生和基因 在下面的前中胚层中表达。 局部化的影响 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 也将被研究。 除了在AER中表达外， 在肢芽的前中胚层中，D1x-5以高量表达， 软骨形成开始时肢芽的近端中央核心 肢体骨骼成分的分化和 几种软骨特异性基质蛋白基因的表达。 D1x-5的表达与乳腺癌的发生也有很强的相关性。 软骨特异性基因的体外表达。 Dix-5表达载体 将用于确定D1x-5是否可以促进以下基因的表达： 软骨特异性基质蛋白，并可调节报告基因的表达 含有II型启动子和增强子元件的构建体 胶原基因 如果是这样的话，D1x-5蛋白与推定的 同源结构域结合序列的第一内含子增强子元件中的同源结构域结合序列。 II型胶原基因在体外和体内的研究。 取决于 根据上述实验的结果，将开始进行研究， 鉴定D1x-5在肢体分化中其他潜在靶基因 D1x-5可能与软骨和蛋白质相互作用以调节基因 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.