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

摘要 生长的骨骼包含称为朗维尔沟（戈尔）的周向解剖结构，其可以 含有骨骼干细胞和对骨骼发育至关重要的信号细胞。临床上，戈尔是重要的 因为涉及戈尔（Salter-Harris II型）的儿科骨折经常影响纵向骨生长。然而，在这方面， 戈尔内调节纵向骨生长的细胞的特性和功能特性并不 知道的我们的初步数据表明，戈尔内的成纤维细胞生长因子受体（FGFRs） 有助于调节相邻生长板软骨细胞和纵向骨生长的“信号中心”。 这一观察结果建立了戈尔和生长板细胞之间的功能联系，并具有一定的意义 儿科骨骺骨折的发病机制我们将这些定义不清的细胞称为“戈尔信号转导 中心”。 用Osx-Cre转基因（Osx-Cre，DCKO小鼠）条件性灭活Fgfr 1和Fgfr 2导致 降低出生后小鼠的纵向骨生长。通过未知的反馈机制， 失活还导致纤维囊和软骨膜中Fgf 9表达增加，这与纤维囊和软骨膜中Fgf 9的表达密切相关。 接近戈尔。在实验上，我们发现软骨膜中FGF 9的表达可以激活 FGFR 3在邻近的增殖软骨细胞中抑制软骨形成。 通过调控Osx-Cre的表达，我们证明Cre在胚胎发育过程中一定是活跃的 来引发这种出生后的生长表型。值得注意的是，Osx-Cre在胚胎期表达，而在出生后不表达， 专门针对戈尔。这些数据在功能上将包围戈尔的区域定义为位置 通过显示胚胎细胞谱系中Fgfr 1/2的失活来研究关键的Fgfr 1/2信号中心， 引起戈尔，而不是在确定的成骨细胞，是负责骨的急剧减少， 在Osx-Cre、DCKO小鼠中观察到生长。 在具体目标1中，我们将使用谱系追踪来差异标记戈尔和单细胞mRNA测序 以鉴定戈尔内具有信号传导中心和/或骨骼干细胞特性的细胞亚群。 这些细胞亚群的选择性标志物的鉴定将用于揭示它们相对于细胞亚群的位置。 解剖学上的戈尔。在具体的目标2中，我们将表征戈尔细胞的信号传导特性，以确定它们的能力。 来调节骨生成和软骨生成。本文提出的研究将确定戈尔的独特特征 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 研究将提供为什么大部分儿童骨骺骨折会导致生长的机理性见解 尽管理论上保留了增殖的软骨细胞和它们完整的血液供应，这些研究 将提供新的遗传工具，研究戈尔在未来的拨款建议和潜在的治疗见解， 可用于预防骨骺骨折引起的生长停滞。

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.