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Characterization of PDGFR dimer-specific dynamics in the craniofacial mesenchyme

颅面间质中 PDGFR 二聚体特异性动力学的表征

基本信息

批准号：
10545287
负责人：
Katherine Ann Fantauzzo
金额：
$ 11.32万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-02 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10545287
关键词：
Ablation Adult Affinity Affinity Chromatography Alleles Binding Biological Assay Branchial arch structure C-terminal Cell Lineage Cell Proliferation Cell physiology Cells Cephalic Cleft Lip Cleft Palate Complement Complex Congenital Abnormality Defect Development Disease Ectoderm Embryo Etiology Fluorescence Fluorescence Microscopy Frozen Sections Gene Expression Genes Genetic Epistasis Genetic Transcription Hemorrhage Human Immunoprecipitation In Vitro Incidence Intracellular Signaling Proteins Knock-in LEOPARD Syndrome Life Ligands Ligation Live Birth Malignant Neoplasms Mass Spectrum Analysis Maxilla Mesenchyme Morphogenesis Mus Mutant Strains Mice N-terminal Nasal septum structure Neural Crest Cell Noonan Syndrome Ontology Output Pattern Phenotype Plasmids Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Play Process Property Protein Dephosphorylation Proteins Proteome Receptor Protein-Tyrosine Kinases Role Signal Transduction Signaling Molecule Techniques Terminator Codon Testing Transcript United States Vascular Diseases Venus Width cohesion craniofacial craniofacial development craniofacial structure differential expression dimer experimental study gain of function mutation homologous recombination in vivo innovation insight migration mutant mutant mouse model nanobodies novel novel therapeutics osteoblast differentiation palatal shelves platelet-derived growth factor BB programs receptor response spatiotemporal transcriptome transcriptome sequencing

项目摘要

Project Summary Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in this process in humans and mice. Pdgfra mutant mouse models display a range of craniofacial phenotypes such as midline clefting, subepidermal blebbing and hemorrhaging. PDGFRa signaling promotes migration of cranial neural crest cells (NCCs), proliferation of the NCC-derived craniofacial mesenchyme and osteoblast differentiation. Recently, a role for PDGFRb has been uncovered in murine craniofacial development, as ablation of Pdgfrb in the NCC lineage results in increased nasal septum width, delayed palatal shelf development and subepidermal blebbing. Further, PDGFRa and PDGFRb have recently been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers. These PDGFRa/b heterodimers have unique signal molecule binding properties and the ability to generate more robust intracellular signaling and mitogenic responses in vitro than those generated by homodimeric receptor complexes. Combined, these findings have shifted the paradigm on how receptor tyrosine kinases act to regulate craniofacial morphogenesis and warrant a full reconsideration of PDGF signaling in midface development. The aim of this proposal is to examine the in vivo dynamics of PDGFR dimer-specific formation, as well as the resulting effects on gene expression and cell activity in the craniofacial mesenchyme. First, PDGFR-bimolecular fluorescence complementation (BiFC) fragment alleles will be generated containing the N- or C-terminal regions of the Venus fluorescent protein. Venus expression will be analyzed in craniofacial structures by fluorescence microscopy to examine the spatiotemporal dynamics of PDGFR homodimer versus heterodimer formation. These alleles will be combined with ectoderm-specific ablation of PDGF-BB ligand to examine the effect on heterodimer formation. Second, the effect of SHP-2 binding to PDGFRa on downstream signaling will be determined through genetic epistasis experiments and, in parallel, BiFC and affinity purification will be employed to selectively purify PDGFRa/b heterodimers and identify PDGFR dimer-specific interacting proteins by mass spectrometry. Finally, RNA-sequencing will be performed to define the transcriptional program induced downstream of PDGFR dimer-specific activation in the maxillary processes. Transcriptional responses involved in proliferation and osteoblast differentiation will be validated through in vivo marker expression analysis to dissect the etiology of the midline defects observed upon ablation of one or both PDGFRs in the NCC lineage. This project will employ innovative techniques to pinpoint the timing and localization of PDGFR dimer-specific activation and analyze the resulting effects on the proteome and transcriptome. These studies will provide significant insight into the mechanisms underlying midface development and new therapeutic directions for the treatment of human craniofacial birth defects.

项目摘要颅面发育是一个复杂的形态发生过程，其中的中断导致高度的普遍存在的人类出生缺陷。通过血小板衍生生长因子受体（PDGFRs）的信号传导发挥作用在人类和小鼠的这一过程中起着关键作用。Pdgfra突变小鼠模型显示一系列颅面表型如中线裂开、表皮下起泡和脱泡。PDGFRa信号传导促进颅神经嵴细胞（NCC）的迁移，NCC衍生的颅面间充质的增殖，成骨细胞分化最近，PDGFRb在小鼠颅面神经中的作用被发现， NCC谱系中Pdgfrb的消融导致鼻中隔宽度增加，腭架发育和表皮下起泡。此外，PDGFRa和PDGFRb最近已被显示在颅面间充质中遗传和物理相互作用以形成功能性异源二聚体。这些PDGFR a/B异二聚体具有独特的信号分子结合特性和产生信号转导的能力。在体外比同二聚体产生的细胞内信号传导和促有丝分裂反应更强受体复合物结合起来，这些发现改变了受体酪氨酸激酶如何作用的范式调节颅面形态发生，并保证全面重新考虑面中部的PDGF信号传导发展该建议的目的是检查PDGFR二聚体特异性形成的体内动力学，以及对颅面间充质中基因表达和细胞活性的影响。第一、 PDGFR-双分子荧光互补（BiFC）片段等位基因将被产生，其含有N- 或者说是维纳斯荧光蛋白的C末端区域。金星表情将在颅面分析通过荧光显微镜检查PDGFR同源二聚体与异二聚体形成。这些等位基因将与PDGF-BB配体的外胚层特异性消融组合，检查对异源二聚体形成的影响。第二，SHP-2与PDGFRa结合对下游细胞的影响。信号传导将通过遗传上位性实验以及平行的BiFC和亲和力测定。纯化将用于选择性纯化PDGFR a/B异二聚体并鉴定PDGFR二聚体特异性相互作用的蛋白质。最后，将进行RNA测序以确定转录程序诱导PDGFR二聚体特异性激活的下游在上颌突。参与增殖和成骨细胞分化的转录反应将通过在体内标记物表达分析，以剖析在消融一个或多个中线缺损后观察到的中线缺损的病因。这两种PDGFR都属于NCC谱系。该项目将采用创新技术来确定时间， PDGFR二聚体特异性激活的定位，并分析对蛋白质组的影响，转录组这些研究将为深入了解面中部的潜在机制提供重要的信息发展和新的治疗方向，为人类颅面出生缺陷的治疗。