Mechanisms of FGFR2 Signaling in Craniofacial Development

描述(申请人提供)：头面部畸形是第四种最常见的先天出生缺陷，发生在新生儿。成纤维细胞生长因子受体2(FGFR2)的显性功能获得突变是人类颅缝融合综合征的主要原因，包括Crouzon、Pfeiffer、Jackson-Weiss、Seathre Chotzen和Apert综合征，这些综合征的特征是在大脑发育完成之前颅缝过早融合。颅缝是一种特殊的关节，含有快速分裂的骨祖细胞和间充质细胞。增殖和分化的成骨细胞之间的平衡受到包括FGFR2在内的生长因子受体的定量信号的精细调节，FGFR2由增殖的成骨细胞表达，在分化的成骨细胞中下调。我们的初步数据显示，FGFR2的突变优先影响神经脊起源的颅骨和面骨。在此，我们提出了两个特定的目标来确定FGFR2信号调控颅面发育和形态发生的机制。在Aim 1.1中，我们建议使用Cre/loxP系统来研究神经脊细胞中突变受体的激活是否足以引起颅突融合综合征，或者它是否需要轴旁中胚层细胞的信号。我们将分别使用Wnt1-Cre或Mesp1-Cre小鼠有条件地激活神经脊细胞或中胚层细胞中的Crouzon突变。一种通用的双重报告菌株将用于从非重组细胞中鉴定重组细胞。缝线将通过组织学、原位杂交和显微CT进行检查。在Aim 1.2中，我们建议使用一种新的双色荧光系统来研究早期成骨细胞和间充质细胞中FGFR2信号的机制。根据成骨分化阶段特异性荧光标记物的表达，通过荧光激活细胞分选分离出三个明确的同质细胞群体，进行基因表达研究。FGFR2突变的细胞内效应和外效应将通过共培养实验进行研究。在目标2中，我们建议确定FGFR2的依赖和独立信号的对接蛋白Frs21，它控制着颅面发育。我们已经证明，将Frs21与突变的FGFR2受体解偶联可以挽救小鼠的颅突融合表型。FGFR2的激活导致Frs21上六个酪氨酸残基的磷酸化，这些残基又作为四个Grb2和两个Shp2信号分子的对接位点。我们的假设是Shp2是FGFR2激活的Frs21病理信号的关键中介。我们将使用两个基因工程Frs21突变小鼠模型来验证这一假设，这两个模型不能在Crouzon突变的背景下招募Grb2或Shp2来响应FGFR的激活。总而言之，这项工作将提供一幅详细的分子图像，说明在正常和疾病条件下，FGFR2和对接蛋白Frs21介导的成纤维细胞生长因子信号如何调节颅面图案和发育。 公共卫生相关性：FGFR2功能突变的显性获得是包括Crouzon、Pfeiffer和Apert综合征在内的大多数人类颅缝融合综合征的原因，这些综合征的特征是在大脑发育完成之前颅缝过早融合。这项提议的目的是获得控制颅面发育的FGFR2刺激所激活的信号通路的全面分子图像。预计这些发现将有助于为FGFRs功能障碍引起的头面部疾病设计新的药理学干预措施。

DESCRIPTION (provided by applicant): Craniofacial anomalies are the fourth most common congenital birth defects that occur in new born. Dominant gain-of-function mutations in the fibroblast growth factor receptor 2 (FGFR2) account for the majority of the human craniosynostosis syndromes including Crouzon, Pfeiffer, Jackson-Weiss, Seathre Chotzen and Apert syndrome which are characterized by the premature fusion of cranial sutures before the completion of brain growth. The cranial sutures are specialized joints which contain rapidly dividing osteoprogenitors and mesenchymal cells. The balance between proliferating and differentiating osteoprogenitors is finely regulated by quantitative signals from growth factor receptors including FGFR2, which is expressed by proliferating osteoprogenitors and down-regulated in differentiating osteoblasts. Our preliminary data show that mutations in FGFR2 preferentially affect cranial and facial bones of neural crest origin. Herein, we propose two Specific Aims to determine the mechanisms of FGFR2 signaling that govern craniofacial development and morphogenesis. In Aim 1.1, we propose to use the Cre/loxP system to investigate whether activation of a mutant receptor in neural crest cells alone is sufficient to cause craniosynostosis syndrome or whether it requires signals from paraxial mesodermal cells. We will conditionally activate the Crouzon mutation in neural crest cells or mesodermal cells using Wnt1-Cre or Mesp1-Cre mice, respectively. A universal dual reporter strain will be used to identify recombinant cells from non-recombinant cells. Sutures will be examined by histology, in situ hybridization and by microCT. In Aim 1.2, we propose to use a novel two color fluorescent system to study the mechanisms of FGFR2 signaling in early osteoprogenitors and mesenchymal cells. Gene expression studies will be performed on three defined homogeneous populations of cells isolated by Fluorescence Activated Cell Sorting based on the expression of osteogenic differentiation stage-specific fluorescent markers. Cell intrinsic versus extrinsic effects of the FGFR2 mutations will be studied by co-culture experiments. In Aim 2, we propose to determine the docking protein Frs21-dependent and independent signaling of FGFR2 that govern craniofacial development. We have shown that uncoupling of Frs21 from the mutant FGFR2 receptor rescues the craniosynostosis phenotype in mice. FGFR2 activation leads to phosphorylation of six tyrosine residues on Frs21, which in turn serve as docking sites for the recruitment of four Grb2 and two Shp2 signaling molecules. Our hypothesis is that Shp2 is the critical mediator of pathological signals of Frs21 from the activated FGFR2. We will test this hypothesis by using two genetically engineered Frs21 mutant mouse models that cannot recruit Grb2 or Shp2 in response to FGFR activation in the context of the Crouzon mutation. Collectively, this work will provide a detailed molecular picture of how FGF-signaling, mediated by the FGFR2 and the docking protein Frs21, regulates craniofacial patterning and development under normal and disease conditions. PUBLIC HEALTH RELEVANCE: Dominant gain of function mutations in FGFR2 accounts for majority of the human craniosynostosis syndromes including Crouzon, Pfeiffer and Apert syndrome which are characterized by the premature fusion of cranial sutures before the completion of brain growth. The goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to FGFR2 stimulation, which govern craniofacial development. It is anticipated that the findings will enable the design of novel pharmacological interventions for craniofacial disorders that are caused by dysfunction in FGFRs.