ROLES OF WNT GENES, HOX GENES, AND AUTOTAXIN IN BONE MORPHOGENESIS

描述：(直接取自用途)分泌的蛋白质指导发育中的骨骼元素的启动、生长和图案形成。该项目将利用逆转录病毒基因转移到发育中的鸡肢芽的强大优势来解决这些步骤中的每一个方面，并将利用该计划提供的转基因核心设施在小鼠身上进行互补的遗传实验。自体趋化蛋白(ATX)是一种分泌型糖蛋白，在软骨形成中心极早期表达。ATX之前已经在人类黑色素瘤细胞的背景下进行了研究，在那里它是一种有效的趋化诱导剂。ATX在发育中的鸡肢芽和微团培养中的错误表达将使其影响软骨形成的能力得到评估。它对肢体间充质细胞迁移的潜在影响将在双室趋化装置中进行检测。间充质凝聚中对ATX的需求将通过在小鼠身上进行定向删除来测试，利用转基因Core。一旦凝结形成，它们的生长就受到许多分泌蛋白质的调节。在肥大的软骨细胞中产生的信号Wnt5b和在形成关节中产生的Wnt14的作用将通过逆转录病毒在发育中的鸡骨中的错误表达来检验。Wnt5b和Wnt14的定向缺失也将与我们的转基因核心一起产生，以确定对这些因素的需求。上位性与IHH和PTHrP的关系将通过检测功能性Wnt5b等位基因的获得和丢失中的基因表达来确定。在我们的转基因核心中，通过引导Wnt5b从ColII启动子转录，Wnt5b也将在小鼠体内过度表达。这些小鼠将与携带IHH和PTHrP突变的小鼠杂交，该项目中的其他项目对此进行了研究，以进一步探索这些信号之间的功能关系。将通过激活形式的FZ受体和显性-阴性形式的β-连环蛋白的错误表达来研究软骨形成过程中的WNT信号转导。虽然所有的骨骼元素都表达相同的信号，但它们会生长到不同的形状和大小。这至少在一定程度上是由HOX基因的差异表达所介导的。携带Hoxa-11和Hoxd-11的靶向缺失或携带表型显性突变ululaless的小鼠，会形成四肢骨骼模式，其中斑脚类前肢的元素，即尺骨和桡骨，无法生长。在这些突变体中，将评估不同信号分子调控的变化。这些纯化因子补充HOX和无尺骨缺陷的能力将在体外培养系统中进行评估。

Description: (Taken directly from the application) Secreted proteins direct the initiation, growth and patterning of the developing skeletal elements. This project will utilize the powerful advantages of retroviral gene transfer into developing chick limb buds to address aspects of each of these steps, and will take advantage of the Transgenic Core facility provided by this Program to carry out complementary genetic experiments in mice. Autotaxin (Atx) is a secreted glycoprotein expressed extremely early in the chondrogenic centers. Atx has been previously studied in the context of human melanoma cells where it is a potent inducer of chemotaxis. Misexpression of Atx in the developing chick limb bud and in micromass culture will allow its ability to affect chondrogenesis to be assessed. Its potential effect on limb mesenchyme migration will be examined in a two-chamber chemotaxis apparatus. The requirement for Atx in mesenchymal condensation will be tested by targeted deletion in mice, utilizing the Transgenic Core. Once condensations have formed their growth is regulated by a number of secreted proteins. The role of Wnt5b, a signal produced in the hypertrophic chondrocytes, and Wnt14, produced in the forming joints, will be examined by retroviral misexpression in developing chick bones. Targeted deletions of Wnt5b and Wnt14 will also be generated in conjunction with our Transgenic Core to determine the requirement for these factors. Epistasis relationships to Ihh and PTHrP will be determined by examining gene expression in the gain and loss of functional Wnt5b alleles. Wnt5b will also be over-expressed by directing its transcription from a ColII promoter in mice in our Transgenic Core. These mice will be crossed with mice carrying mutations in Ihh and PTHrP, studied in other projects in this Program, to further explore the functional relationships between these signals. Wnt signal transduction will be studied during cartilage formation by misexpression of activated forms of Fz receptors and dominant-negative form of beta-catenin. While all the skeletal elements express the same signals, they grow to distinct shapes and sizes. This is, at least in part, mediated by differential expression of Hox genes. Mice carrying targeted deletions of both Hoxa-11 and Hoxd-11, or harboring the phenocopy dominant mutation ulnaless, form skeletal limb patterns where the zeugopod forelimb elements, the radius and ulna, fail to grow. Changes in regulation of various signaling molecules will be assessed in these mutants. The ability of these purified factors to complement the Hox and ulnaless defects will be assessed in an in vitro culture system.