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。PDGFRA突变小鼠模型显示一系列头面部 表型如中线裂开、真皮下起泡和出血。PDGFRA信号促进 颅神经嵴细胞迁移、颅面间充质细胞增殖及细胞分化 成骨细胞分化。最近，PDGFRb在小鼠颅面中的作用被发现 发展，因为PDGFRb在NCC谱系中的消融导致鼻中隔宽度的增加，延迟 腭架发育和真皮下起泡。此外，PDGFRA和PDGFRb最近被 显示了在基因和物理上相互作用的头面部间充质形成功能异二聚体。 这些PDGFRA/b异源二聚体具有独特的信号分子结合特性和产生 体外细胞内信号和有丝分裂反应比同源二聚体更强 受体复合体。总而言之，这些发现改变了受体酪氨酸激酶的作用模式。 调节颅面形态发生并充分考虑面中部的PDGF信号转导 发展。这项建议的目的是研究PDGFR二聚体特异性形成的体内动力学， 以及由此对颅面间充质中基因表达和细胞活性的影响。第一, 将产生PDGFR-双分子荧光互补(BIFC)片段等位基因，包含N- 或金星荧光蛋白的C末端区域。金星的表情将在颅面进行分析 用荧光显微镜观察PDGFR同源二聚体与 杂二聚体的形成。这些等位基因将结合外胚层特异性的PDGF-BB配体的消融来 考察其对杂二聚体形成的影响。第二，SHP-2与PDGFRA结合对下游的影响 信号将通过遗传上位性实验以及BIFC和亲和力来确定 将采用纯化的方法选择性地纯化PDGFRA/b杂二聚体并鉴定PDGFR二聚体 通过质谱学分析相互作用的蛋白质。最后，将进行RNA测序以确定 转录程序在上颌突中诱导PDGFR二聚体特异性下游激活。 参与增殖和成骨细胞分化的转录反应将通过 活体标记物表达分析用于解剖消融一个或多个中线缺陷的病因 两个PDGFR都在NCC谱系中。该项目将采用创新的技术来确定时间和 PDGFR二聚体特异性激活的定位及其对蛋白质组和蛋白质组的影响 转录组。这些研究将对面中部潜在的机制提供重要的见解。 人类头面部出生缺陷治疗的发展和新的治疗方向。

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.