Identification of an FGF-regulated signaling center in the Groove of Ranvier that controls longitudinal bone growth.

摘要 正在生长的骨骼包含一种名为兰维尔沟(GOR)的周向解剖结构，它可能 含有骨骼干细胞和对骨骼发育至关重要的信号细胞。在临床上，GOR是重要的 因为累及GOR(Salter-Harris II型)的儿童骨折通常会影响纵向骨生长。然而， GOR内调节纵向骨生长的细胞的特性和功能特性尚不清楚 为人所知。我们的初步数据表明，大脑皮层内成纤维细胞生长因子受体(FGFRs) 有助于形成一个“信号中心”，调节邻近的生长板软骨细胞和纵向骨生长。 这一观察结果在大脑皮层和生长板的细胞之间建立了功能联系，并具有一定的意义 儿童骨盆骨折的发病机制。我们将这些定义不清的细胞称为“GOR信号 中心“。 OSX-CRE转基因(OSX-CRE，DCKO小鼠)条件失活FGFR1和FGFR2导致 减少出生后小鼠的纵向骨骼生长。通过一个未知的反馈机制，这 失活还会导致纤维被膜和软骨膜中Fgf9表达增加，这一点与此密切相关 离戈尔很近。实验上，我们发现FGF9在软骨膜中的表达可以激活 FGFR3在邻近增殖的软骨细胞中抑制软骨形成。 通过调节OSX-Cre的表达，我们证明了Cre在胚胎发育过程中必须是活跃的 以诱导这种出生后的生长表型。显著地，OSX-Cre在胚胎而不是出生后表达 专门针对GOR。这些数据在功能上将围绕GOR的区域定义为位置 通过显示FGFR1/2在胚胎细胞谱系中失活，从而揭示了关键的FGFR1/2信号中心 导致骨骼急剧减少的是GOR，而不是最终的成骨细胞 在OSX-CRE，DCKO小鼠中观察到生长。 在特定的目标1中，我们将使用谱系追踪来区别标记GOR和单细胞mRNA测序 以确定GOR内具有信号中心和/或骨骼干细胞特性的细胞亚群。 识别这些细胞亚群的选择标记将被用来揭示它们相对于 解剖上的GOR。在具体目标2中，我们将表征GOR细胞的信号特性，以了解它们的能力 调节成骨和软骨生成。这里提出的研究将确定GOR的独特特征 细胞作为潜在的细胞信号中心，具有调节邻近生长板组织的独特能力。这些 研究将提供机理上的洞察，以解释为什么大部分儿童骨干骨骨折会导致生长。 尽管理论上保留了增殖的软骨细胞及其完整的血液供应，但仍被阻止。这些研究 将在未来的拨款提案和潜在的治疗洞察力中提供新的遗传工具来研究GOR 可以用来防止与生长板骨折相关的生长停滞。

ABSTRACT Growing bones contain a circumferential anatomical structure called the groove of Ranvier (GOR) that may contain skeletal stem cells and signaling cells critical to skeletal development. Clinically, the GOR is important as pediatric fractures involving the GOR (Salter-Harris type II) often affect longitudinal bone growth. However, the identity and functional properties of cells within the GOR that regulate longitudinal bone growth are not known. Our preliminary data suggest that Fibroblast Growth Factor Receptors (FGFRs) within the GOR contribute to a “signaling center” that regulates adjacent growth plate chondrocytes and longitudinal bone growth. This observation establishes a functional link between cells in the GOR and the growth plate and has implications for the pathogenesis of pediatric physeal fractures. We refer to these poorly defined cells as the “GOR signaling center”. Conditional inactivation of Fgfr1 and Fgfr2 with the Osx-Cre transgene (Osx-Cre, DCKO mice) results in decreased longitudinal bone growth in postnatal mice. Through an unknown feedback mechanism, this inactivation also results in increased Fgf9 expression in the fibrous capsule and perichondrium, which is in close proximity to the GOR. Experimentally, we showed that expression of FGF9 in the perichondrium can activate FGFR3 in adjacent proliferating chondrocytes to suppress chondrogenesis. By regulating the expression of Osx-Cre, we show that Cre must be active during embryonic development to elicit this postnatal growth phenotype. Significantly, embryonic, but not postnatal, expression of Osx-Cre specifically targets the GOR. These data functionally define the region encompassing the GOR as the location of a critical FGFR1/2 signaling center by showing that inactivation of Fgfr1/2 in an embryonic cell lineage that gives rise to the GOR, rather than in definitive osteoblasts, is responsible for the dramatic reduction in bone growth observed in Osx-Cre, DCKO mice. In specific aim 1, we will use lineage tracing to differentially label the GOR and single cell mRNA sequencing to identify cell sub-populations within the GOR that have signaling center and/or skeletal stem cell properties. Identification of selective markers for these cell sub-populations will be used to reveal their location relative to the anatomical GOR. In specific aim 2, we will characterize the signaling properties of GOR cells for their ability to regulate osteogenesis and chondrogenesis. The studies proposed here will identify unique features of GOR cells as a potential cell signaling center with the unique ability to regulate adjacent growth plate tissue. These studies will provide mechanistic insight into why a large percentage of pediatric physeal fractures result in growth arrest despite theoretically preserving the proliferating chondrocytes and their intact blood supply. These studies will provide new genetic tools to study the GOR in future grant proposals and potential therapeutic insight that could be used to prevent growth arrest associated with physeal fractures.