PDGFRbeta signaling in craniofacial development.

描述(申请人提供)：颅面发育是一个复杂的形态发生过程，其中的干扰导致非常普遍的出生缺陷在人类。通过血小板衍生生长因子受体α(PDGFRα)传递的信号在颅面部发育中起着关键作用；该基因的突变小鼠模型显示出面部裂隙、真皮下起泡和面部出血等表型。虽然该家族中的另一种受体酪氨酸激酶β在颅面发育中的作用尚未确定，但有限的文献证据表明，这两种受体可能能够形成具有与同源二聚体受体复合体不同性质的功能异二聚体。我们的初步体外研究表明，PDGFRα和PDGFRβ在原代培养的小鼠胚胎腭间充质细胞中表达，并能够形成具有激活下游信号通路能力的功能异二聚体。进一步的体内初步分析表明，在颅面发育过程中，这两种受体在神经脊细胞谱系中相互作用，因为双纯合子突变胚胎表现出明显的面部劈裂表型，其真皮下气泡比任何一个突变胚胎中观察到的更严重。这项应用的目的是探索PDGFRβ在头面部发育中的作用，长期目标是识别在中线发育过程中该受体下游工作的新效应分子，并表征它们在面部裂隙和真皮下起泡的病因学中的作用。首先，将使用同源重组敲入方法来产生PdgfrbmCherry空报告等位基因。记者表情会 使用荧光体视显微镜对整个头面部结构进行分析，并在冠状冰冻切片上进行免疫荧光分析。其次，通过Western blotting、BrdU掺入实验、细胞生长曲线和Transwell实验分别评估PDGFRβ同源二聚体下游和PDGFRα/β异源二聚体激活对信号通路的激活、增殖、存活和迁移的影响。最后，在活体颅面发育过程中，将通过使用两个基因的条件等位基因和WNT1-Cre驱动程序来靶向消融神经脊，来表征PDGFRα和PDGFRβ的相互作用。通过形态、组织学和标记表达分析，将复合突变小鼠的表型与单一纯合子突变胚胎的表型进行比较。本申请中概述的研究策略将利用PDGF信号通路可用的大量试剂，以及产生重要的新工具，以探索颅面生物学的新方面。本文提出的创新性研究将探索PDGFRβ在面部间充质中的表达，并确定在颅面发育过程中PDGFFRβ同源二聚体和PDGFFRα/β异二聚体激活下游的信号通路和细胞过程，最终为预防颅面出生缺陷提供新的治疗方向。

DESCRIPTION (provided by applicant): Craniofacial development is a complex morphogenic process, disruptions in which result in highly prevalent birth defects in humans. Signaling through platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development; mutant mouse models of the gene display phenotypes such as facial clefting, subepidermal blebbing and facial hemorrhaging. While the other receptor tyrosine kinase in the family, PDGFRβ, does not have an established role in craniofacial development, limited evidence from the literature indicates that the two receptors may be able to form functional heterodimers with distinct properties from homodimeric receptor complexes. Our preliminary in vitro studies reveal that PDGFRα and PDGFRβ are expressed in primary mouse embryonic palatal mesenchyme (MEPM) cells and are capable of forming functional heterodimers with the capacity to activate downstream signaling pathways. Further preliminary in vivo analyses demonstrate that the two receptors interact in the neural crest cell lineage during craniofacial development, as double homozygous mutant embryos exhibit an overt facial clefting phenotype with subepidermal blebbing that is more severe than that observed in either single mutant embryo. The aim of this application is to explore the role of PDGFRβ in craniofacial development, with a long-term goal of identifying novel effector molecules operating downstream of this receptor during midline development and characterizing their role in the etiology of facial clefting and subepidermal blebbing. First, a homologous recombination knock- in approach will be used to generate a PdgfrbmCherry null reporter allele. Reporter expression will be analyzed in craniofacial structures in whole mount using a fluorescent stereomicroscope and in coronal frozen sections by immunofluorescence analysis. Second, the activation of signaling pathways, proliferation, survival and migration will be assessed downstream of PDGFRβ homodimer versus PDGFRα/β heterodimer activation by Western blotting, BrdU incorporation assays, cell growth curves and Transwell assays, respectively. Finally, the interaction of PDGFRα and PDGFRβ will be characterized during craniofacial development in vivo, by employing conditional alleles for both genes together with a Wnt1-Cre driver to target ablation in the neural crest. The phenotypes of compound mutant mice will be compared to those of single homozygous mutant embryos by morphological, histological and marker expression analyses. The research strategy outlined in this application will take advantage of the vast array of reagents available for the PDGF signaling pathway, as well as generate vital new tools, to explore novel aspects of craniofacial biology. The innovative studies proposed here will explore the expression of PDGFRβ in the facial mesenchyme and identify the signaling pathways and cellular processes induced downstream of PDGFRβ homodimer and PDGFRα/β heterodimer activation during craniofacial development, ultimately providing new therapeutic directions aimed at the prevention of craniofacial birth defects.