Mechanisms of FGFR2 Signaling in Craniofacial Development

FGFR2 信号在颅面发育中的机制

基本信息

批准号：
8415478
负责人：
JACOB V.P. ESWARAKUMAR
金额：
$ 39.21万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-03 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8415478
关键词：
Accounting Affect Alanine Animal Model Animals Apert syndrome Apert-Crouzon syndrome Binding Sites Biochemical Biological Models Birth Brain Breeding Cells Cephalic Chotzen Syndrome Cleft Palate Coculture Techniques Color Congenital Abnormality Craniofacial Abnormalities Craniosynostosis Data Defect Development Disease Docking Dysplasia EphA4 Receptor Ephrin-A2 Equilibrium FGFR2 gene Fibroblast Growth Factor Fibroblast Growth Factor Receptor 2 Fibroblast Growth Factor Receptors Fluorescence-Activated Cell Sorting Functional disorder Gene Expression Gene Targeting Genetic Genetic Engineering Genetically Engineered Mouse Germ Lines Goals Growth Growth Factor Receptors Histology Human In Situ Hybridization In Vitro Intervention Joint structure of suture of skull Joints Knock-in Mouse Life Link Mediating Mediator of activation protein Mesenchymal Mesenchyme Mesoderm Cell Methods Molecular Molecular Analysis Morphogenesis Mus Mutant Strains Mice Mutate Mutation Neural Crest Neural Crest Cell Osteoblasts Pathway interactions Pattern Pfeiffer Syndrome Phenotype Phosphorylation Phosphotransferases Population Prevalence Proliferating Protein Isoforms Protein Tyrosine Phosphatase Proteins Recombinants Recruitment Activity Reporter Role Signal Pathway Signal Transduction Signaling Molecule Site Specificity Staging Staining method Stains Stem cells Surgical sutures Syndrome System Testing Tyrosine Work base bone coronal suture craniofacial cranium design face bone structure fibroblast growth factor receptor 2c gain of function gain of function mutation genome-wide loss of function mRNA Expression mutant mutant mouse model neuronal cell body novel osteogenic osteoprogenitor cell premature public health relevance receptor research study response stem suture fusion

项目摘要

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.

描述（由申请人提供）：颅面异常是新生中发生的第四个最常见的先天性先天出生缺陷。成纤维细胞生长因子受体2（FGFR2）中的主要功能突变占大多数人类颅骨突变综合征，包括Crouzon，Pfeiffer，Pfeiffer，Jackson-Weiss，Seathre Chotzen和Apert综合征，这些综合征是由脑神经促成过早促进的大脑生长的过早融合的表征。颅骨缝合线是专门的关节，其中包含迅速分裂的骨基因生成剂和间充质细胞。增殖和分化的骨基因生成剂之间的平衡受到来自包括FGFR2在内的生长因子受体的定量信号来调节，FGFR2通过增生的骨基因生成剂表达并在分化成骨细胞中下调。我们的初步数据表明，FGFR2中的突变优先影响神经rest起源的颅骨和面部骨骼。在本文中，我们提出了两个特定的目的，以确定控制颅面发育和形态发生的FGFR2信号的机制。在AIM 1.1中，我们建议使用CRE/LOXP系统来研究单独的神经rest细胞中突变受体的激活是否足以引起颅突式综合征，或者是否需要从偏式中胚层细胞中的信号。我们将分别使用WNT1-CRE或MESP1-CRE小鼠有条件地激活神经Crest细胞或中胚层细胞中的Crouzon突变。通用双报告菌株将用于鉴定来自非重组细胞的重组细胞。缝合线将通过组织学，原位杂交和Microct进行检查。在AIM 1.2中，我们建议使用一种新型的两种颜色荧光系统来研究早期骨化剂和间充质细胞中FGFR2信号的机制。基因表达研究将基于成骨分化阶段特异性荧光标记物的表达，对三个定义的细胞均匀群体进行基因研究。通过共培养实验，将研究FGFR2突变的细胞固有与外在效应。在AIM 2中，我们建议确定控制颅面发育的FGFR2的对接蛋白FRS21依赖性和独立的信号传导。我们已经表明，FRS21与突变体FGFR2受体的解偶联可挽救小鼠中的颅骨表型。 FGFR2激活导致FRS21上六个酪氨酸残基的磷酸化，这又是募集四个GRB2和两个SHP2信号分子的对接位点。我们的假设是SHP2是激活FGFR2的FRS21病理信号的关键介体。我们将通过使用两个基因工程的FRS21突变小鼠模型来检验该假设，这些模型无法募集GRB2或SHP2在Crouzon突变的背景下响应FGFR激活。总的来说，这项工作将提供详细的分子图片，说明FGF-Signaling如何通过FGFR2和对接蛋白FRS21介导的FGF信号如何调节正常和疾病条件下的颅面模式和发育。