Growth Factor Signaling and Craniofacial Development

描述（由申请人提供）：本提案的主要目的是确定由PDGF信号控制的中面发育机制。PDGFRa或其配体PDGFA和PDGFC的缺失会导致面部裂和额鼻突发育不正常，而该通路的亚形态突变会导致腭裂。本申请提出：1。表征PDGFRa在颅神经嵴细胞和颅面发育中调控的过程。PDGFRa信号的缺失影响两个不同的过程，额鼻肿块的发育和中线的融合。我们将评估颅神经嵴细胞在PDGFRa条件和半形突变体中填充额鼻肿块的能力，使用体内Cre谱系追踪和体外趋化试验以及颅神经嵴细胞外植体。我们还将详细检查额鼻肿块的发展和中线融合。2. 研究PDGF下游参与PI3K/AKT信号通路在颅面发育中的作用。我们之前的遗传实验已经确定了PI3K信号通路在颅面发育过程中介导PDGF活性的核心作用，但进一步的下游通路仍然未知。我们将通过质谱鉴定原代腭间质细胞中PI3K/AKT的磷酸化靶点。将进行PDGFRa和磷酸化靶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. 建立指导颅面PDGF信号下游模式的遗传途径。我们已经在原代腭间充质细胞中发现了许多PDGF的转录靶点，这些靶点参与了额鼻过程和腭的细胞迁移和模式规范。选择这些PDGF靶基因将用于分离小鼠突变体。表型分析将在零或条件靶基因突变中进行，以确定额鼻过程和上颚的变化。与PDGFRa突变体的遗传相互作用将被执行，以建立在颅面发育中PDGF下游的遗传途径。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上进行了精细的分析，并且由于这些详细的知识提供的见解，预计提出的研究将对颅面生物学产生重大影响。这一建议将为颅面出生缺陷的预防开辟新的方向。

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.