Characterization of PDGFR dimer-specific dynamics in the craniofacial mesenchyme

项目摘要 颅面发育是一个复杂的形态发生过程，其中的干扰会导致高度 普遍存在的人类先天缺陷。通过血小板衍生生长因子受体(PDGFRs)传递信号 在人类和小鼠的这一过程中起着关键作用。PDGFRpha和PDGFRBeta最近被证明 遗传和物理上相互作用的头面部间充质形成具有独特功能的异二聚体 信号分子结合特性和产生更强大的细胞内信号和有丝分裂的能力 体外反应比由同源二聚体受体复合体产生的反应更好。这项建议的目的是 检测PDGFR二聚体特异性形成的体内动力学及其对基因的影响 颅面间充质细胞的表达和细胞活性。第一，PDGFR-双分子荧光 将产生包含N端或C端区域的互补(BIFC)片段等位基因 金星荧光蛋白。金星在颅面结构中的表达将用荧光分析 显微镜检查PDGFR同源二聚体与异源二聚体形成的时空动力学。 其次，SHP-2与PDGFRpha结合对下游信号转导的影响将通过基因决定。 上位性实验以及BIFC和亲和纯化将被用于选择性纯化 PDGFRpha/Beta异二聚体，并通过质谱学鉴定PDGFR二聚体特异性相互作用蛋白。 最后，将进行RNA测序以确定在其下游诱导的转录程序 PDGFR二聚体在上颌突中的特异性激活。其他实验、技术和培训 在此独立科学家奖的申请中引入了显著扩展和 加强原有的研究计划，并为职业发展留出余地。其中包括细胞生物学和超分辨。 显微镜训练以表征细胞内的亚细胞定位和内化动力学 各种PDGFR二聚体；应用生物信息学方法进行RNA测序分析的培训； 以及参加领导力和科学沟通培训计划。这项培训计划将在 在科罗拉多大学安舒茨医学院牙科医学院，它有一个 强大的、成熟的颅面生物学研究计划，并提供充足的培训机会 和协作。大学支持包括超过奖励期限的有保证的工资支持；提供 用于博士后招募、共享设备使用、课程学费和到科学学院的旅费的资金 会议；允许候选人基本上全职进行研究；以及参与 旨在促进候选人研究发展的联合实验室会议和工作组 程序。综合起来，这些活动将有助于实现建立公认的研究的目标。 实验室致力于了解支配哺乳动物头面部的信号等级 以及这些信号事件的中断如何导致人类头面部出生缺陷。

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.