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

描述：（直接取自应用程序）分泌的蛋白质指导骨骼元素发育的起始、生长和模式。该项目将利用逆转录病毒基因转移到小鸡肢体芽发育中的强大优势，解决上述每个步骤的各个方面，并将利用该项目提供的转基因核心设施，在小鼠身上进行补充基因实验。Autotaxin （Atx）是一种在软骨形成中心极早表达的分泌糖蛋白。Atx先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质量培养中的错误表达将使其影响软骨形成的能力得到评估。其对肢体间质迁移的潜在影响将在双腔趋化装置中进行检查。我们将利用转基因核心，通过小鼠的靶向删除来测试间充质凝聚对Atx的需求。凝析物一旦形成，它们的生长就受到一些分泌蛋白的调节。Wnt5b（一种产生于肥大软骨细胞的信号）和Wnt14（一种产生于形成关节的信号）的作用将通过在发育中的鸡骨骼中逆转录病毒的错误表达来检验。Wnt5b和Wnt14的靶向缺失也将与我们的转基因核心一起产生，以确定对这些因子的需求。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来确定。在我们的转基因核心中，Wnt5b也会通过引导其从大肠杆菌启动子转录而在小鼠中过度表达。这些小鼠将与本项目其他项目中携带Ihh和PTHrP突变的小鼠杂交，以进一步探索这些信号之间的功能关系。Wnt信号转导将在软骨形成过程中通过Fz受体的激活形式和β -连环蛋白的显性阴性形式的错误表达进行研究。虽然所有的骨骼元素都表达相同的信号，但它们会长成不同的形状和大小。这至少部分是由Hox基因的差异表达介导的。携带Hoxa-11和Hoxd-11靶向缺失的小鼠，或无表型显性突变的小鼠，形成骨肢模式，其中舟足类前肢元素，桡骨和尺骨不能生长。各种信号分子调控的变化将在这些突变体中进行评估。这些纯化因子补充Hox和非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.