FGF signaling during growth and mechanical adaptation of tendon-bone interfaces

摘要 肌腱-骨界面是一种结构上分级的纤维软骨界面，对转导至关重要。 从肌肉到骨骼的机械载荷。肌肉负荷减少会导致界面生长受损 和骨骼畸形。最近，我们已经证明了胚胎中肌腱-骨骼界面的大小 小鼠肢体受成纤维细胞生长因子9(FGF9)和骨骼肌特异性基因敲除的负调控 FGF9的表型复制效果与我们在全球FGF9突变体中看到的效果相同。这些强有力的初步发现使我们 假设成纤维细胞生长因子信号，由肌肉特异性的FGF9介导，有助于非 细胞自主方式。然而，目前还不清楚成纤维细胞生长因子是否以及如何在相邻肌肉之间传递信号， 肌腱和骨骼改变界面祖细胞的行为，或者界面生长是否次要于 成纤维细胞生长因子介导从肌肉到机械负荷的变化。在这份提案中，我们将使用创新鼠标 组织特异性CRE中成纤维细胞生长因子配体和受体功能丧失(LOF)和功能获得(GOF)的模型 等位基因来分离成纤维细胞生长因子诱导的变化所需的细胞群体。我们将严格比较结构上的 (即，微型计算机断层扫描)、细胞/ECM(即，组织形态学)和生物力学特性(即， LOF/GOF基因型和对照的肌腱-骨界面的拉伸、压痕试验)。我们将测试FGFR- 化学性肌肉卸载后界面依赖的机械反应性结构适应 使用光遗传刺激去神经或增加肌肉负荷。在这个项目中，我们将使用创新的、 利用脉冲蓝光诱导新生小鼠重复骨骼肌收缩的活体光遗传学 我们在最近的NIH R03支持下建立的技术。该方法与新型FGFR LOF/GOF相结合 小鼠模型，将使我们能够确定成纤维细胞生长因子信号在肌肉负荷诱导的适应中的作用 肌腱-骨骼界面。我们的目标是(1)证明肌源性Fgf9负责调节 肌腱-骨界面在胚胎和出生后发育过程中的发育及(2)机制的建立 通过这种细胞特异性的成纤维细胞生长因子信号调节负荷诱导的肌腱-骨界面的结构适应。 这一R01提案将确立Fgf9在界面生长和 肌腱-骨界面的结构/功能及FGFR信号在界面中的细胞自主作用 生长和机械适应。此外，这项工作的发现将确定保守的和独特的 引导腱-骨出生后生长的与成纤维细胞生长因子受体相关的下游信号通路 接口。

ABSTRACT The tendon-bone interface is a structurally graded fibrocartilage interface that is critical for transducing mechanical loads from muscle to the skeleton. Reduced muscle loading can lead to impaired interface growth and skeletal deformities. Recently, we have shown that the size of tendon-bone interfaces in the embryonic mouse limb are negatively regulated by fibroblast growth factor 9 (FGF9), and skeletal muscle-specific knockout of FGF9 phenocopies the effects we see in global FGF9 mutants. These strong preliminary findings have led us to hypothesize that FGF signaling, mediated by muscle specific FGF9, contributes to interface growth in a non- cell autonomous manner. However, it remains unclear if and how FGF signaling between adjacent muscle, tendon, and bone alters the behavior of interface progenitor cells, or whether interface growth is secondary to FGF-mediated changes to mechanical loading from muscle. In this proposal, we will use innovative mouse models for FGF ligand- and receptor-loss of function (LOF) and gain-of-function (GOF) in tissue-specific Cre alleles to isolate the cell population required for FGF-induced changes. We will rigorously compare the structural (i.e., microcomputed tomography), cellular/ECM (i.e., histomorphology) and biomechanical properties (i.e., tensile, indentation tests) of the tendon-bone interface in LOF/GOF genotypes and controls. We will test FGFR- dependent mechanoresponsive structural adaptation of interfaces following muscle unloading using chemical denervation or increased muscle loading using optogenetic stimulation. In this project, we will use innovative, in vivo optogenetics to induce repetitive skeletal muscle contraction in neonatal mice using pulsed blue light, a technique we established with recent NIH R03 support. This approach, combined with novel FGFR LOF/GOF mouse models, will allow us to determine the role of FGF signaling in muscle loading induced adaptation of the tendon-bone interface. We aim to (1) demonstrate muscle-derived Fgf9 is responsible for regulating the development of tendon-bone interfaces during embryonic and postnatal growth and (2) Establish the mechanism by which cell-specific FGF signaling regulates loading-induced structural adaptations of tendon-bone interfaces. This R01 proposal will establish the non-cell autonomous contributions of Fgf9 in interface growth and structure/function of tendon-bone interfaces as well as the cell autonomous roles of FGFR signaling in interface growth and mechanical adaptation. Additionally, findings from this work will identify both conserved and unique downstream signaling pathways associated with FGF receptors that guide postnatal growth of tendon-bone interfaces.