Growth Factor Signaling and Craniofacial Development

描述(由申请人提供)：这项建议的主要目的是确定由PDGF信号控制的面中部发育的潜在机制。PDGFRA或其配体PDGFA和PDGFC的缺失会导致面部裂隙和额鼻突的不正常发育，而这一通路的亚型突变会导致腭裂。本研究的目的是：1.研究PDGFRA对颅神经嵴细胞和颅面发育的调控作用。PDGFRA信号的丢失影响两个不同的过程，额鼻区肿块的发展和中线的融合。我们将使用体内CRE谱系追踪、体外趋化试验和颅神经脊细胞外植体来评估PDGFRA条件突变体和亚型突变体中颅神经脊细胞在额鼻部肿块中的填充能力。我们还将详细检查额鼻肿块和中线融合的发展情况。2.探讨PDGF下游参与PI3K/AKT信号转导通路在颅面部发育中的作用。我们之前的遗传学实验已经指定PI3K信号通路在调节PDGF在颅面发育中的活性中起核心作用，但更多的下游通路仍不清楚。我们将通过质谱学鉴定PI3K/AKT在原代腭间充质细胞中的磷酸化靶点。将进行PDGFRA和磷酸化目标突变体之间的遗传上位性实验，以确定它们在颅面发育中的作用。3.建立一条指导PDGF信号下游颅面构型的遗传途径。我们已经在原始的腭间充质细胞中确定了一些PDGF的转录靶点，它们参与了额鼻突和腭部的细胞迁移和模式指定。选择这些PDGF靶基因将用于分离小鼠突变体。将对无效或有条件的靶基因突变体进行表型分析，以确定额鼻突和腭部的变化。将进行与PDGFRA突变体的遗传相互作用，以建立在颅面发育中PDGF下游操作的遗传途径。在生长因子信号通路中，PDGF信号通路已经在分子和细胞水平得到了细致的分析，由于这一详细知识提供的洞察力，拟议中的研究有望对颅面生物学产生重大影响。因此，这项建议将为预防头面部出生缺陷开辟新的方向。

DESCRIPTION (provided by applicant): The major aims of this proposal are to identify mechanisms underlying midface development that are controlled by PDGF signaling. Loss of the PDGFRa or of its ligands PDGFA and PDGFC lead to facial clefting and improper development of the frontonasal process, whereas hypomorphic mutations in this pathway result in cleft palate. This application proposes: 1. To characterize the processes regulated by PDGFRa in cranial neural crest cells and craniofacial development. Loss of PDGFRa signaling affects two distinct processes, the development of the frontonasal masses and fusion at the midline. We will assess the ability of cranial neural crest cells to populate the frontonasal mass in PDGFRa conditional and hypomorphic mutants, using in vivo Cre lineage tracing and in vitro chemotaxis assays and cranial neural crest cell explants. We will also examine in detail the development of the frontonasal masses and midline fusion. 2. To investigate the pathways that operate downstream of PDGF engaged PI3K/AKT signaling in craniofacial development. Our previous genetic experiments have assigned a central role to the PI3K signaling pathway in mediating PDGF activity in craniofacial development, but further downstream pathways remain unknown. We will identify by mass spectroscopy the phosphorylation targets of PI3K/AKT in primary palatal mesenchyme cells. Genetic epistasis experiments between PDGFRa and phosphorylation target mutants will be conducted to identify their role in craniofacial development. 3. To establish a genetic pathway that instructs craniofacial patterning downstream of PDGF signaling. We have identified a number of transcriptional targets of PDGF in primary palatal mesenchymal cells, which are involved in cell migration and pattern specification in the frontonasal process and the palate. A selection of these PDGF target genes will be used to isolate mouse mutants. Phenotyping will be performed in null or conditional target gene mutants to determine changes in the frontonasal process and the palate. Genetic interactions with PDGFRa mutants will be performed to establish a genetic pathway operating downstream of PDGF in craniofacial development. Among growth factor signaling pathways, PDGF signaling has been exquisitely analyzed at a molecular and cellular level, and the proposed studies are anticipated to have significant impact in craniofacial biology because of the insight provided by this detailed knowledge. This proposal will thus open new directions for the prevention of craniofacial birth defects.