Mechanisms of FGFR2 Signaling in Craniofacial Development

描述（由申请人提供）：颅面畸形是新生儿中第四常见的先天性出生缺陷。成纤维细胞生长因子受体2（FGFR 2）中的显性功能获得性突变导致了大多数人类颅缝早闭综合征，包括Crouzon、Pfeiffer、Jackson-Weiss、Seathre Chotzen和Apert综合征，其特征在于脑生长完成前颅缝的过早融合。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。增殖和分化的骨祖细胞之间的平衡由来自生长因子受体（包括FGFR 2）的定量信号精细调节，所述生长因子受体由增殖的骨祖细胞表达并且在分化的成骨细胞中下调。我们的初步数据表明，FGFR 2的突变优先影响神经嵴起源的颅骨和面骨。在此，我们提出了两个具体的目标，以确定机制的FGFR 2信号调控颅面发育和形态发生。在目的1.1中，我们建议使用Cre/loxP系统，以调查是否激活神经嵴细胞中的突变型受体单独是足以导致颅缝早闭综合征或是否需要从近轴中胚层细胞的信号。我们将分别使用Wnt 1-Cre或Mesp 1-Cre小鼠在神经嵴细胞或中胚层细胞中有条件地激活Crouzon突变。通用双报告菌株将用于从非重组细胞中鉴别重组细胞。将通过组织学、原位杂交和microCT检查缝线。在目的1.2中，我们提出使用一种新的双色荧光系统来研究早期骨祖细胞和间充质细胞中FGFR 2信号转导的机制。基于成骨分化阶段特异性荧光标记物的表达，将对通过荧光激活细胞分选法分离的三个确定的同质细胞群进行基因表达研究。将通过共培养实验研究FGFR 2突变的细胞内在效应与外在效应。在目的2中，我们提出确定对接蛋白Frs 21依赖和独立的信号FGFR 2支配颅面发育。我们已经证明，Frs 21从突变型FGFR 2受体解偶联挽救了小鼠的颅缝早闭表型。FGFR 2激活导致Frs 21上6个酪氨酸残基的磷酸化，这反过来又作为4个Grb 2和2个Shp 2信号分子募集的对接位点。我们的假设是，Shp 2是从激活的FGFR 2的病理信号的Frs 21的关键介质。我们将通过使用两种基因工程Frs 21突变小鼠模型来测试这一假设，这两种小鼠模型在Crouzon突变的背景下不能招募Grb 2或Shp 2来响应FGFR激活。总的来说，这项工作将提供一个详细的分子图片FGF信号，介导的FGFR 2和对接蛋白Frs 21，调节颅面图案和正常和疾病条件下的发展。

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.