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

WNT 基因、HOX 基因和自分泌因子在骨形态发生中的作用

• 批准号: 6395870
• 负责人: CLIFFORD J. TABIN
• 金额: $ 17.51万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6395870
• 关键词: biological signal transduction; bone development; cadherins; cartilage development; chemotaxis; chick embryo; developmental genetics; gene deletion mutation; gene targeting; homeobox genes; laboratory mouse; limbs; mesenchyme; osteogenesis; secretory protein; tissue /cell culture; transfection /expression vector

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.

描述：（直接取自应用程序）分泌的蛋白质直接启动，生长和发育骨骼元素的模式。该项目将利用逆转录病毒基因转移到发育中的鸡肢芽中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中进行补充遗传实验。自分泌运动因子（Atx）是一种分泌型糖蛋白，在软骨形成中心极早表达。Atx先前已在人类黑色素瘤细胞的背景下进行了研究，其中它是一种有效的趋化性诱导剂。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。其对肢体间充质迁移的潜在影响将在双室趋化装置中进行检查。将利用转基因核心，通过小鼠中的靶向缺失来测试间充质凝聚中对Atx的需求。一旦凝聚体形成，它们的生长就受到许多分泌蛋白的调节。Wnt 5 b的作用，在肥大的软骨细胞中产生的信号，和Wnt 14，在形成关节中产生的，将通过逆转录病毒在发育中的鸡骨中的错误表达来检查。Wnt 5 b和Wnt 14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。通过检查功能性Wnt 5 b等位基因获得和丧失中的基因表达，确定与Ihh和PTHrP的上位关系。在我们的转基因核心中，Wnt 5 b也将通过指导其在小鼠中从ColII启动子转录而过表达。这些小鼠将与携带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期间通过Fz受体的活化形式和β-连环蛋白的显性负性形式的错误表达来研究。虽然所有的骨骼元素都表达相同的信号，但它们会生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，或携带表型显性突变ulnaless的小鼠，形成骨骼肢体模式，其中zeugopod前肢元素，桡骨和尺骨，无法生长。将在这些突变体中评估各种信号分子调节的变化。将在体外培养系统中评估这些纯化因子补充Hox和无尺骨缺陷的能力。