PDGFRbeta signaling in craniofacial development.

描述（由申请人提供）：颅面发育是一个复杂的形态形成过程，其中断导致人类高度普遍的出生缺陷。通过血小板衍生生长因子受体α (PDGFRα)的信号传导在颅面发育中起关键作用；该基因的突变小鼠模型表现出面部裂裂、表皮下起泡和面部出血等表型。虽然家族中的另一种受体酪氨酸激酶PDGFRβ在颅面发育中没有确定的作用，但文献中有限的证据表明，这两种受体可能能够形成与同型二聚体受体复合物具有不同性质的功能性异二聚体。我们的初步体外研究表明，PDGFRα和PDGFRβ在小鼠胚胎腭间质（MEPM）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性异源二聚体。进一步的初步体内分析表明，在颅面发育过程中，两种受体在神经嵴细胞系中相互作用，因为双纯合突变胚胎表现出明显的面部裂型，伴有表皮下起泡，比单突变胚胎更严重。本应用的目的是探索PDGFRβ在颅面发育中的作用，其长期目标是在中线发育过程中识别在该受体下游操作的新型效应分子，并描述其在面部裂和皮下水泡病因学中的作用。首先，采用同源重组敲入方法生成PdgfrbmCherry空报告等位基因。记者表达将

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.