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信号控制的面中部发育的潜在机制。PDGFRa或其配体PDGFA和PDGFC的缺失导致面部裂开和额鼻突的不适当发育，而该途径中的亚形态突变导致腭裂。本申请提出：1.描述PDGFRa在颅神经嵴细胞和颅面发育中的调控过程。PDGFRa信号传导的缺失影响两个不同的过程，即额鼻肿块的发育和中线融合。我们将使用体内Cre谱系追踪和体外趋化性测定以及颅神经嵴细胞外植体来评估颅神经嵴细胞在PDGFRa条件突变体和亚型突变体中填充额鼻肿块的能力。我们还将详细检查额鼻肿块的发展和中线融合。2.研究PDGF参与的PI 3 K/AKT信号通路在颅面发育中的下游作用。我们先前的遗传实验已经将PI 3 K信号通路在颅面发育中介导PDGF活性中的中心作用分配给PI 3 K信号通路，但是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充质细胞中PI 3 K/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鉴定它们在颅面发育中的作用。3.建立一个遗传途径，指导颅面模式下游的血小板衍生生长因子信号。我们已经确定了一些转录的PDGF在原代腭间充质细胞，这是参与细胞迁移和模式规范在额鼻突和腭的目标。这些PDGF靶基因的选择将用于分离小鼠突变体。将在无效或条件性靶基因突变体中进行表型分析，以确定额鼻突和腭的变化。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。在生长因子信号传导途径中，PDGF信号传导已经在分子和细胞水平上进行了精细的分析，并且由于这种详细的知识所提供的洞察力，预计所提出的研究将对颅面生物学产生重大影响。因此，这项建议将为预防颅面出生缺陷开辟新的方向。 公共卫生相关性：包括唇腭裂在内的颅面发育性疾病是全球人口中最常见的出生缺陷。这项建议的主要目的是确定由PDGF信号控制的机制，这些机制是面中部正常发育的基础。我们将研究PDGF如何控制神经嵴细胞迁移，识别参与中线发育的下游细胞内信号通路，并表征介导这些过程的转录靶点。

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. PUBLIC HEALTH RELEVANCE: Craniofacial developmental diseases including cleft lip or palate are the most prevalent birth defects in the human population worldwide. The major aims of this proposal are to identify mechanisms controlled by PDGF signaling that underlie normal development of the midface. We will investigate how PDGF controls neural crest cell migration, identify downstream intracellular signaling pathways involved in midline development, and characterize the transcriptional targets that mediate these processes.