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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：
10576282
负责人：
Katherine Ann Fantauzzo
金额：
$ 12.53万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10576282
关键词：
Ablation Adult Affinity Affinity Chromatography Alleles Authorization documentation Award Binding Bioinformatics Biological Assay Biology Branchial arch structure Bulla C-terminal Cell Lineage Cell physiology Cells Cellular biology Cephalic Cleft Lip Cleft Palate Collaborations Colorado Communication Complement Complex Congenital Abnormality Defect Dental Schools Development Disease Ectoderm Embryo Equipment Etiology Event Fluorescence Fluorescence Microscopy Frozen Sections Funding Gene Expression Genes Genetic Epistasis Genetic Transcription Goals Hemorrhage Human Immunoprecipitation In Vitro Incidence Independent Scientist Award Intracellular Signaling Proteins Joints Knock-in LEOPARD Syndrome Laboratory Research Leadership Life Ligands Ligation Live Birth Malignant Neoplasms Mass Spectrum Analysis Maxilla Medical Medicine Mesenchyme Molecular 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 Platelet-Derived Growth Factor alpha Receptor Platelet-Derived Growth Factor beta Receptor Play Postdoctoral Fellow Process Proliferating Property Protein Dephosphorylation Proteins Proteome Receptor Protein-Tyrosine Kinases Research Role Signal Transduction Signaling Molecule Techniques Technology Terminator Codon Testing Training Training Programs Transcript Travel United States Universities Vascular Diseases Venus Wages Width career development craniofacial craniofacial development craniofacial structure design differential expression dimer experimental study gain of function mutation homologous recombination in vivo innovation insight loss of function meetings migration mutant mutant mouse model nanobodies novel novel therapeutics osteoblast differentiation palatal shelves platelet-derived growth factor BB programs receptor recruit response spatiotemporal superresolution microscopy training opportunity transcriptome transcriptome sequencing working group

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. PDGFRalpha and PDGFRBeta have recently been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers with 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. 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. Second, the effect of SHP-2 binding to PDGFRalpha on downstream signaling will be determined through genetic epistasis experiments and, in parallel, BiFC and affinity purification will be employed to selectively purify PDGFRalpha/Beta 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. Additional experiments, technologies and training opportunities are introduced in this Independent Scientist Award application that significantly extend and enhance the original research plan and allow for career development. These include cell biology and superresolution microscopy training to characterize the subcellular localization and internalization dynamics of the various PDGFR dimers; training in the application of bioinformatics approaches for RNA-sequencing analysis; and participation in leadership and scientific communication training programs. This training plan will take place within the School of Dental Medicine at the University of Colorado Anschutz Medical Campus, which has a strong, well-established research program in craniofacial biology and provides ample opportunities for training and collaboration. University support includes guaranteed salary support beyond the award period; provision of funds for post-doctoral fellow recruitment, shared equipment usage, course tuition and travel to scientific meetings; permission for the candidate to spend essentially full-time conducting research; and participation in joint lab meetings and working groups designed to facilitate the development of the candidate's research program. Combined, these activities will contribute towards the goal of establishing a recognized research laboratory committed to understanding the signaling hierarchies that govern mammalian craniofacial development and how disruption of these signaling events leads to human craniofacial birth defects.

项目总结