GENETIC ANALYSIS OF CHONDROGENESIS

描述(由申请者提供)：本项目的目标是确定Sox5和Sox6在从妊娠晚期到成年的软骨形成和维持中的作用。这些基因编码类似的Sry相关转录因子，以前被证明在小鼠胚胎的软骨形成中具有必要的、多余的作用。它们通过在多个步骤控制软骨细胞分化，促进软骨内骨软骨模板的形成和生长板的建立。由于决定这些功能的常规突变体在出生前或出生后不久死亡，Sox5/Sox6在妊娠晚期的一些作用尚不清楚，出生后的作用也完全未知。虽然在胚胎中获得的数据以及胚胎和出生后软骨之间的强烈相似性表明，在以后的生命中具有重要的功能，但需要新的实验来验证这一假设。该项目将专门确定SOX5/SOX6是否需要在妊娠晚期和新生儿期形成关节和关节软骨，以及生长板和关节软骨在整个成年期的维持。在后一种情况下，关节软骨细胞不表达Sox5/Sox6被认为是导致骨关节炎的原因。基于Sox5/Sox6在决定软骨细胞命运中的作用，本项目还将测试Sox5/Sox6除了先前已发现的细胞外基质基因外，是否还控制调控基因的表达。为了绕过早期致死，目标1是使用Cre-loxP系统产生携带Sox5/Sox6条件等位基因的小鼠系。目的2是将这些小鼠与已建立的Cre转基因小鼠结合起来，在软骨细胞或软骨形成部位的亚群中特异性地灭活Sox5/Sox6，以研究从胚胎发育到成年的功能。目的3是利用已建立的Cre小鼠或产生新的Cre小鼠，以时间调节的方式灭活Sox5/Sox6，从而研究不依赖于胚胎功能的出生后功能。建议进行组织学和分子分析来确定突变小鼠的特征。目的4利用基因表达谱芯片和体内定量分析获得软骨细胞中Sox5/Sox6相关基因的详细表达谱。这项研究将大大扩展我们对重要的软骨形成转录因子的作用的认识，并将为控制关节形成、软骨生长板的维持、关节软骨形成和关节骨性退行性变的分子机制提供新的见解。

DESCRIPTION (provided by applicant): The goal of this project is to identify the roles of Sox5 and Sox6 in cartilage formation and maintenance from late gestation onto adulthood. The genes encode similar Sry-related transcription factors and were previously demonstrated to have essential, redundant roles in chondrogenesis in the mouse embryo. They promote formation of the cartilage templates of endochondral bones and establishment of growth plates by controlling chondrocyte differentiation at multiple steps. Since the conventional mutants generated to determine these functions die before or soon after birth, some of the roles of Sox5/Sox6 in late gestation are unknown and postnatal roles remain totally unknown. While the data obtained in embryos and strong similarities between embryonic and postnatal cartilages suggest important functions later in life, new experiments are needed to test this hypothesis. This project will specifically determine whether Sox5/Sox6 are needed for joint and articular cartilage formation, which occur in late gestation and the neonatal period, and for growth plate and articular cartilage maintenance throughout adulthood. In the latter case, failure to express Sox5/Sox6 in articular chondrocytes is hypothesized to lead to osteoarthritis. Based on the roles of Sox5/Sox6 in determining chondrocyte fate, this project will also test whether Sox5/Sox6 control expression of regulatory genes besides the extracellular matrix genes previously identified. To bypass early lethality, Aim 1 is to use the Cre-loxP system to generate mouse lines harboring Sox5/Sox6 conditional alleles. Aim 2 is to use these mice in conjunction with established Cre transgenic mice to inactivate Sox5/Sox6 specifically in chondrocytes or in subsets of chondrogenic sites to study functions from embryogenesis through adulthood. Aim 3 is to use established Cre mice or generate new ones to inactivate Sox5/Sox6 in a temporally regulated manner and thereby study postnatal functions independently of embryonic functions. Histological and molecular analyses are proposed to characterize mutant mice. Aim 4 is to use gene expression arrays and quantitative in vivo assays to obtain a detailed profile of Sox5/Sox6-related gene expression in chondrocytes. This study should significantly extend our knowledge of the roles of important chondrogenic transcription factors and should provide novel insights into the molecular mechanisms that control joint formation, maintenance of cartilage growth plate, articular cartilage formation, and osteoarthritic degeneration of articular joints.