Role of Ossifying Chondrocytes in Regeneration of the Adult Jaw Skeleton

项目总结/摘要 人类健康的一个主要目标是提高大骨折和骨骼伤口愈合的能力。相比之下 对于哺乳动物来说，许多两栖动物和蜥蜴确实具有改革整个肢体和/或尾巴的非凡能力 骨骼，但这些物种相对缺乏遗传工具，限制了对潜在的 细胞和分子机制。在这里，我们提出了一个新的模型，骨骼再生的基因， 温顺的斑马鱼在短短几周内，成年斑马鱼可以再生近三分之二的细胞。 他们似乎是通过一种不寻常的软骨细胞群来做到这一点的， 编织骨由于在哺乳动物骨折修复过程中观察到了潜在的相似细胞， 了解这些细胞在骨骼修复，以及它们如何有助于更广泛的 在低等脊椎动物中的骨再生，将有助于开发用于改善患者骨修复的新疗法。 在第一个目的中，再生软骨细胞的纯化和表达谱分析，所述再生软骨细胞表达以下标志物： 软骨细胞和成骨细胞，将决定这些细胞是杂交软骨细胞的程度。 成骨细胞在早期再生软骨细胞中特异性上调的基因也将表明潜在的 诱导这些细胞对损伤做出反应的途径。接下来，我们使用新开发的Cre/Lox转基因品系， 来测试再生软骨细胞的起源和长期命运。特别是，我们测试骨膜是 再生软骨细胞的主要来源，这些细胞直接转化为成骨细胞， 编织骨使用一种新的交叉转基因策略来特异性消融再生软骨细胞， 然后我们测试这些细胞是斑马鱼颌骨大规模骨再生所必需的。 在软骨内骨的发育过程中，大多数软骨细胞经历肥大， 细胞凋亡，骨基质由侵入的成骨细胞产生。在再生过程中， 初步数据表明许多软骨细胞直接分化成成骨细胞。利用一个成年的 ihha突变体和转基因策略，以抑制Hh信号仅在再生软骨细胞，我们测试了在 第二个目的是持续高的Ihh信号传导对于再生软骨细胞分化成 成骨细胞这些目标的完成将测试一个模型，即再生软骨细胞的能力， 直接制造骨允许在受损的身体部分中快速恢复刚性， 然后被重塑成成熟的骨头从长远来看，我们计划从这种新的斑马鱼身上吸取教训， 模型来设计策略，以增强骨骼修复临界尺寸缺陷的固有能力。 !

Project Summary/Abstract A major goal in human health is to improve the ability of large fractures and skeletal wounds to heal. In contrast to mammals, many amphibia and lizards do have a remarkable ability to reform entire limb and/or tail skeletons, yet the relative lack of genetic tools in these species have limited progress towards the underlying cellular and molecular mechanisms. Here, we present a new model of skeletal regeneration in the genetically tractable zebrafish. In a matter of just a few weeks, adult zebrafish can regenerate nearly two-thirds of their lower jawbone, and they appear to do so through an unusual chondrocyte population that directly produces woven bone. As potentially similar cells have been observed during mammalian fracture repair, a better understanding of these cells during skeletal repair, as well as how they contribute to more extensive regeneration in lower vertebrates, will aid in developing novel therapies for improving bone repair in patients. In the first aim, purification and expression profiling of regenerating chondrocytes, which express markers of both chondrocytes and osteoblasts, will determine the extent to which these cells are hybrid chondrocytes/ osteoblasts. Genes specifically upregulated in early regenerating chondrocytes will also indicate potential pathways that induce these cells in response to injury. Next, we use newly developed Cre/Lox transgenic lines to test the origins and long-term fate of regenerating chondrocytes. In particular, we test that the periosteum is a major source of regenerating chondrocytes, with these directly converting into the osteoblasts that produce woven bone. Using a novel intersectional transgenic strategy to specifically ablate regenerating chondrocytes, we then test that these cells are required for the large-scale regeneration of bone in the zebrafish jaw. During the development of endochondral bone, the majority of chondrocytes undergo hypertrophy and apoptosis, with bony matrix being produced by invading osteoblasts. Quite differently during regeneration, our preliminary data suggest that many chondrocytes directly differentiate into osteoblasts. Using an adult viable ihha mutant and a transgenic strategy to inhibit Hh signaling only in regenerating chondrocytes, we test in the second aim that persistently high Ihh signaling is essential for regenerating chondrocytes to differentiate into osteoblasts. The completion of these Aims will test a model that the ability of regenerating chondrocytes to directly make bone allows a rapid restoration of rigidity in a damaged body part, with the initial woven bone later being remodeled into mature bone. In the long-term, we plan to use lessons taken from this new zebrafish model to devise strategies to augment the inherent ability of the skeleton to repair critical size defects. !