Feedback Inhibitory Mechanisms in Skeletal Development

描述（由申请人提供）：成纤维细胞生长因子（FGFs）和成纤维细胞生长因子受体（fgfr）在发育中起重要作用，包括骨骼和颅面发育。人类FGFR1、FGFR2和FGFRS的激活突变与包括颅缝闭锁和侏儒综合征在内的骨骼发育不良有关。这些突变影响成骨细胞和软骨细胞的增殖和分化。这一数据表明FGFR信号传导在骨骼发育中的关键作用：FGFR信号传导必须严格调节才能正常发育。尚未解决的重要问题是骨骼发育过程中fgfr激活的通路的识别，以及这些通路如何反馈调节FGF信号传导。对果蝇和脊椎动物的研究表明，Spry基因家族的成员是FGFR信号传导的抑制剂。在小鼠发育过程中，Spry1、Spry2和Spry4在肢体芽、上颌和下颌弓以及其他部位表达。逆转录病毒介导的Spry1在鸡肢体芽中的过度表达可导致软骨发育不良。我们开发了一种条件转基因小鼠模型来探索Spry家族成员在骨骼发育中的作用。我们发现，Spry1在颅神经嵴细胞中的条件表达会导致严重的颅面缺损，包括鼻骨和额骨的缺失。这些小鼠颅面原基中转录因子Msx1、Msx2和AP2的表达也大大降低。我们假设Spry在骨骼发育中的功能是维持fgf介导的增殖、分化和凋亡之间的平衡，而Spry的表达水平决定了这种平衡。因此，我们提出以下3个具体目的：1)验证Spry过表达抑制骨原基FGF活性，导致整体成骨细胞发育下降的假设；2)利用功能缺失方法研究Spry在体内成骨细胞增殖、分化和凋亡中的作用；3)利用体外颅骨培养物表征Spry影响成骨细胞增殖、分化和凋亡的机制。这些研究将为颅面和骨骼发育中信号通路的负调控以及这种反馈信号通路的扰动如何导致骨骼发育不良提供重要的见解。

DESCRIPTION (provided by applicant): The fibroblast growth factors (FGFs) and the fibroblast growth factor receptors (FGFRs) play important roles in development, including skeletal and craniofacial development. Activating mutations in human FGFR1, FGFR2, and FGFRS are associated with skeletal dysplasias including craniosynostosis and dwarfing syndromes. These mutations affect the proliferation and differentiation of osteoblasts and chondrocytes. This data indicates a pivotal role for FGFR signaling in skeletal development: signaling by FGFRs must be tightly regulated for normal development. Important unresolved issues are the identification of pathways activated by FGFRs during skeletal development, and how these pathways feedback to regulate FGF signaling. Studies in Drosophila and vertebrates have demonstrated that members of the Sprouty (Spry) gene family are inhibitors of FGFR signaling. Spry1, Spry2, and Spry4 are expressed in the limb buds, and maxillary and mandibular arches, as well as other sites during mouse development. Retroviral-mediated over-expression of Spry1 in chick limb buds results in chondrodysplasia. We have developed a conditional transgenic mouse model to explore the role of Spry family members in skeletal development. We show that conditional expression of Spry1 in cranial neural crest cells results in severe craniofacial defects including the absence of the nasal and frontal bones. These mice also exhibit greatly reduced expression of the transcription factors Msx1, Msx2, and AP2 in craniofacial primordia. We hypothesize that the function of Spry in skeletal development is to maintain a balance between FGF-mediated proliferation, differentiation, and apoptosis, and that the level of Spry expression determines this balance. Accordingly, we propose the following 3 specific aims: 1) to test the hypothesis that over- expression of Spry inhibits FGF activity in bone primordia, leading to decreased overall osteoblast development; 2) to use loss-of-function approaches to investigate the role of Spry in osteoblast proliferation, differentiation, and apoptosis in vivo; and 3) to characterize the mechanisms by which Spry affects osteoblast proliferation, differentiation, and apoptosis using calvarial cultures in vitro. These studies will provide significant insight into the negative regulation of signaling pathways in craniofacial and skeletal development and how perturbations in this feedback signaling pathway lead to skeletal dysplasias.