Mechanisms of Synovial Joint Formation

 描述（由申请人提供）：滑膜关节对于骨骼功能至关重要，并且对其结构、不同组织以及对先天性和获得性疾病（包括骨关节炎（OA））的易感性了解很多。相反，人们对它们的发育生物学知之甚少。如果这些信息可用，它可以直接使用或与生物工程工具一起使用，以创建新的关节修复策略。这个项目开始了好几年 之前就填补了这些重要的空白。在肢体中，关节形成起始于软骨性长骨原基内的每个预期关节部位处的间充质细胞间带的出现。然而，长期以来，人们一直不清楚区间细胞是否作为一个短暂的关节分界点，它们是否真的随着时间的推移产生关节组织，如果是的话，它们如何执行这一关键任务。为了解决这些问题，我们使用Gdf 5基因追踪了区间细胞，发现Gdf 5-Cre/R26谱系细胞随着时间的推移持续存在并产生关节组织，因此代表了具有关节形成能力的特化祖细胞。我们还发现Wnt/β- catenin信号传导对于表达Prg 4/润滑素的表面区细胞是重要的。尽管这些数据很新颖，但仍有很多问题没有得到解答，包括Gdf 5谱系细胞如何产生不同的关节组织，关节软骨如何生长并获得重要的细胞柱状组织， 联合祖细胞是否在出生后持续存在，以及它们在修复中可能扮演什么角色。为了解决这些问题，我们创建了新的Gdf 5-CreER和Prg 4-CreER系，并使用了包括Axin 2-CreER在内的其他系。我们的初步数据现在表明：（a）产前关节内的祖细胞被顺序指定为对不同组织的贡献;（b）生长中的长骨骺上的初始关节软骨的侧向扩张涉及Ranvier沟附近的祖细胞，具有Axin 2谱系;（B）（c）关节软骨成熟和柱状组织化涉及细胞嵌入机制，而不是如其他人所建议的贴壁生长;和（iv）Kartogenin增强关节祖细胞性能和Prg 4表达。这些数据导致了我们的中心假设，即关节形成是由胚胎发生过程中顺序指定的祖细胞引起的模块化过程，Wnt/β-连环蛋白代表了主要的调控途径。我们还假设，出生后关节软骨的生长和成熟需要软骨细胞结构和表型的特定变化，关节细胞的修复能力是服从药理学或遗传刺激。我们的目标是：（1）在产前发育和产后生长期间对关节祖细胞进行命运定位;（2）确定关节软骨增厚并获得其柱状组织的机制;以及（3）评估关节祖细胞及其后代的修复能力以及在骨骼和/或遗传上促进其修复的方法。该项目将对关节形成和修复的机制产生全新的见解，因此具有相当大的基础和转化医学相关性和意义。

 DESCRIPTION (provided by applicant): Synovial joints are essential for skeletal function, and much is known about their structure, distinct tissues and susceptibility to congenital and acquired diseases, including osteoarthritis (OA). In contrast, little is known about their developmental biology. Were such information available, it could be used directly or together with bioengineering tools to create new joint repair strategies. This project started several years ago to fill these important gaps. In the limb, joint formation initiates with appearance of a mesenchymal cell interzone at each prospective joint site within the cartilaginous long bone anlagen. However, it had long remained unclear whether the interzone cells serve as a transient joint demarcation point, whether they actually produce joint tissues over time and if so, how they perform that key task. To address these questions, we genetically tracked interzone cells using Gdf5 and found that Gdf5-Cre/R26-lineage cells persisted and produced joint tissues over time, thus representing specialized progenitors with joint formation capacity. We found also that Wnt/β- catenin signaling was important for surface zone cells expressing Prg4/lubricin. Though novel, the data left much unanswered, including how the Gdf5-lineage cells produce different joint tissues, how articular cartilage grows and acquires its important cell columnar organization, whether joint progenitors persist postnatally, and what roles they could have in repair. To address these issues, we created new Gdf5-CreER and Prg4-CreER lines and used others including Axin 2-CreER. Our preliminary data now indicate that: (a) progenitors within the prenatal joint are sequentially specified for contribution to different tissues; (b) lateral expanson of incipient articular cartilage over the growing long bone epiphysis involves progenitors near the groove of Ranvier with an Axin 2-lineage; (c) articular cartilage maturation and columnar organization involve cell intercalation mechanisms rather than appositional growth as suggested by others; and (iv) joint progenitor performance and Prg4 expression are enhanced by Kartogenin. These data lead to our central hypothesis that joint formation is a modular process brought about by progenitors specified sequentially during embryogenesis, with Wnt/β-catenin representing a major regulatory pathway. We also hypothesize that postnatal articular cartilage growth and maturation require specific changes in chondrocyte cytostructure and phenotype, and that joint cells' repair capacity is amenable to pharmacological or genetic stimulation. Our Aims are: (1) To fate-map joint progenitors during prenatal development and postnatal growth; (2) To determine mechanisms by which articular cartilage thickens and attains its columnar organization; and (3) To assess the repair capacity of joint progenitors and their progenies and means to boost it pharmacologically and/or genetically. The project will produce fundamentally new insights into the mechanisms of joint formation and repair and will thus have considerable basic and translational medicine relevance and significance.