Novel Effectors of FGF Signaling in Craniofacial Development

颅面发育中 FGF 信号传导的新型效应器

• 批准号: 9909681
• 负责人: James Clark
• 金额: $ 6.61万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909681
• 关键词: Adaptor Signaling Protein; Address; Alleles; Amino Acids; Binding; Binding Proteins; Biochemical Genetics; Biological Assay; Birth; Cell Culture Techniques; Cell Proliferation; Cell physiology; Cells; Cessation of life; Cleft lip with or without cleft palate; Complex; Congenital Abnormality; Congenital Disorders; Coupled; Craniofacial Abnormalities; Development; Developmental Process; Disease; Embryo; Epitopes; Exhibits; FGFR2 gene; Family; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Genes; Genetic; Human; Immunoprecipitation; In Vitro; Individual; Ligands; Literature; LoxP-flanked allele; MAPK3 gene; Mass Spectrum Analysis; Mediating; Mesenchyme; Morphogenesis; Mus; Mutation; Neural Crest; Output; Pathway interactions; Phenocopy; Phenotype; Phosphotransferases; Play; Prevention; Prevention approach; Process; Proteins; Proteomics; Reagent; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Research; Role; Series; Signal Pathway; Signal Transduction; Stable Isotope Labeling; Syndrome; Techniques; Terminator Codon; Therapeutic; Therapeutic Intervention; Tissues; Work; combinatorial; craniofacial; craniofacial development; craniofacial disorder; experimental study; genetic analysis; growth factor receptor-bound protein 2; in vivo; innovation; insight; member; migration; mutant; novel; novel therapeutic intervention; null mutation; progenitor; receptor; receptor binding; recruit; src-Family Kinases

Project Summary The Fibroblast Growth Factor (FGF) signaling pathway consists of eighteen FGF ligands and four FGF receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family. FGF signaling is integral to mammalian development and is active throughout numerous tissues and stages in the developing embryo. Additionally, FGF signaling is a major coordinator of craniofacial morphogenesis. Misregulation of FGF is implicated in a wide range of congenital disorders, such as cleft lip or palate (CL/P). Numerous studies have examined the role of individual ligand-receptor combinations necessary for different aspects of development, as well as the various intracellular signaling pathways that are activated downstream of FGF signaling. FGFRs have been shown to recruit various adaptor proteins that facilitate the propagation of intracellular signals such as the ERK1/2 and PI3K pathways. Previous work by the Soriano lab has shown that FGF controls craniofacial development through the combinatorial recruitment of various effectors that activate multiple downstream signaling pathways. However, there are still gaps in the complete picture of how FGF signaling is relayed downstream of receptor activation, as abrogating the binding of known effectors does not replicate the phenotype of Fgfr null mutations. The aim of this proposal is to identify these missing effectors. I will utilize two complementary approaches to identify novel effectors of FGF signaling: a hypothesis-driven candidate approach and an unbiased protein screen through stable isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry (MS). To analyze the potential roles of these effectors, I will utilize in vivo and in vitro techniques to assay genetic interactions and cellular functions. I will quantify cellular proliferation, death, and migration, and changes in downstream signaling dynamics, both in the presence of wild-type or signaling mutant backgrounds. From those targets that exhibit positive evidence of downstream interaction with FGFRs, we will select critical genes for further biochemical and genetic analysis. Utilizing a neural crest specific Cre line, Wnt1Cre2, we will examine the role of each novel effector in craniofacial development on their own, or in conjunction with Fgfr null or signaling mutant backgrounds to verify in vivo genetic interaction. The innovative studies proposed here aim to provide novel insight into the mechanism by which FGFR activation propagates intracellular signals to coordinate the complexity of craniofacial development. Our research strategy will utilize an array of existing reagents, alongside newly developed strains, to uncover missing aspects of FGF signaling and provide new avenues for therapeutic intervention and prevention of congenital craniofacial disorders.

项目摘要 成纤维细胞生长因子（FGF）信号传导途径由18个FGF配体和4个组成 FGF受体（FGFR），受体酪氨酸激酶（RTK）家族的成员。 FGF信号传导与 哺乳动物发育，并且在发育中的胚胎中遍及整个组织和各个阶段。 另外，FGF信号传导是颅面形态发生的主要协调员。 fgf的不利位置是 与多种先天性疾病有关，例如唇裂或pa（Cl/p）。有许多研究 研究了开发不同方面所必需的单个配体受体组合的作用， 以及FGF信号下游激活的各种细胞内信号通路。 FGFR已显示可募集各种辅助蛋白，以促进细胞内传播 诸如ERK1/2和PI3K途径之类的信号。 Soriano Lab的先前工作表明FGF 通过组合募集各种效应子来控制颅面的发育 多个下游信号通路。但是，关于FGF的完整情况，仍然存在差距 信号传导是受体激活下游的中继，因为废除已知效应子的结合不 复制FGFR无效突变的表型。该提案的目的是确定这些缺失的效应子。 我将利用两种互补方法来识别FGF信号的新型效果：假设驱动的 候选方法和通过氨基酸在细胞中稳定的同位素标记的无偏蛋白筛选 培养（SILAC）与质谱法（MS）结合。为了分析这些效应子的潜在作用，我将 利用体内和体外技术来分析遗传相互作用和细胞功能。我会量化 细胞增殖，死亡和迁移，以及下游信号传导动力学的变化，都在 存在野生型或信号突变体背景。从那些表现出积极证据的目标 与FGFR的下游相互作用，我们将选择关键基因以进行进一步的生化和遗传 分析。利用神经CREST特定的CRE线Wnt1cre2，我们将检查每个新型效应器的作用 在颅面开发中，或与FGFR无效或信号突变体背景结合使用 验证体内遗传相互作用。这里提出的创新研究旨在提供对 FGFR激活传播细胞内信号以协调复杂性的机制 颅面发展。我们的研究策略将利用一系列现有试剂以及新的 开发的菌株，以发现FGF信号的缺失方面并为治疗提供新的途径 干预和预防先天性颅面疾病。