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：
10358047
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 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10358047
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

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

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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，干扰导致高度普遍的人类先天缺陷。通过血小板衍生的生长因子受体（PDGFR）发出信号传导在这一过程中的关键作用在人类和小鼠中。 PDGFRA突变鼠标模型显示了一系列颅面表型，例如中线裂，皮下注射和出血。 PDGFRA信号促进颅神经rest细胞（NCC）的迁移，NCC衍生的颅面间充质的增殖和成骨细胞分化。最近，PDGFRB的角色在鼠颅颅中发现了发展，随着NCC谱系中PDGFRB的消融导致鼻中隔宽度增加，延迟帕拉特架子的发展和皮下注射。此外，PDGFRA和PDGFRB最近在颅面间充质中以遗传和物理相互作用以形成功能性异二聚体。这些PDGFRA/B异二聚体具有独特的信号分子结合特性和产生的能力比同二聚体产生的细胞内信号传导和有丝分裂反应的体外更强受体复合物。这些发现结合在一起，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在中间完全重新考虑PDGF信号发展。该建议的目的是检查PDGFR二聚体特异性形成的体内动力学，以及对颅面间充质基因表达和细胞活性产生的影响。第一的， PDGFR分子荧光互补（BIFC）片段将产生，其中包含N- 金星荧光蛋白的C末端区域。金星表达将在颅面中进行分析通过荧光显微镜进行结构，以检查PDGFR同型二聚体的时空动力学异二聚体形成。这些等位基因将与PDGF-BB配体的外胚层特异性消融结合到检查对异二聚体形成的影响。其次，SHP-2与PDGFRA结合对下游的影响信号传导将通过遗传性位解实验确定，并并行，BIFC和亲和力纯化将用于选择性纯化PDGFRA/B异二聚体并识别PDGFR二聚体特异性通过质谱法相互作用的蛋白质。最后，将执行RNA测序以定义转录程序在上颌过程中引起了PDGFR二聚体特异性激活的下游。将通过IN验证与增生和成骨细胞分化有关的转录反应体内标记表达分析是为了剖析一个或一个中线缺陷的病因 NCC谱系中的两个PDGFR。该项目将采用创新技术来指出时间和 PDGFR二聚体特异性激活的定位，并分析对蛋白质组和转录组。这些研究将对中层的机制提供重大洞察力用于治疗人类颅面先天缺陷的新的治疗方向。