Regulation of upper lip development by PDGFR Alpha andRac1 signaling

描述(由申请人提供)：唇裂是最常见的出生缺陷之一，全世界每700名新生儿中就有1名患有唇裂。这是由于环境或遗传因素破坏了正常的上唇发育。这项研究的长期目标是了解上唇发育的机制和口裂的发病机制。在人类和小鼠的唇腭裂中，血小板衍生生长因子受体α(PDGFRα)信号的突变与唇腭裂密切相关，这表明在颅面发育中具有进化保守的作用。胚胎发育过程中，内侧鼻突(MNP)、外侧鼻突(LNP)和上颌突(MxP)的高度协调发育形成上唇，它们都起源于神经脊细胞(NCC)。我以前的研究表明，PDGFRα信号是维持细胞增殖和调节NCC迁移所必需的，并表明小GTP酶Rac1在PDGFRα下游的这些过程中发挥作用。本研究旨在研究RAC1信号在介导PDGFRα调控硝普钠和鼻咽癌发生中的作用。在第一个目标中，我将使用新产生的无偏倚的WNT1-Cre2等位基因，特异性地去除NCC中的rac1功能，并在组织学、细胞和分子水平上表征突变的头面部表型。Rac1和PDGFRα之间的上位性效应将通过灭活NCC中每个基因的单个等位基因来测试。我会进一步挽救头面部的表型 Pdgfrαfl/fl；wnt1-cre2小鼠通过在体内驱动构成活性形式的rac1在NCC中的表达。在第二个目标中，我将研究在α形态发生过程中PDGFRAC1信号是如何调节RAC1活性的。我将分析PDGFRα缺陷的MNP的直接下游信号通路的活性，并研究已发现的信号通路与RAC1活性之间的相互作用。目的研究人牙周膜生长因子受体α转录靶标CDC42ep3的表达及其在颅面部发育中的作用。CEP3将通过体外检测和基因打靶的方法进行检测。这些工作的结果将在信号转导和转录水平上描绘PDGFRα调控NCC和MNP发育的信号级联。本研究的结果将为理解MNP发育和上唇形态发生的基本机制提供新的信息。这项研究将对唇裂的治疗具有重要意义，最终减少这种出生缺陷在人类中的发生。

DESCRIPTION (provided by applicant): Cleft lip represents one of most common birth defects and affects approximately 1 in every 700 newborns worldwide. It is caused by disruption of normal upper lip development with environmental or genetic factors. The long term goal of this proposed research is to understand the mechanisms of upper lip development and of orofacial cleft pathogenesis. Mutations of Platelet Derived Growth Factor Receptor α (PDGFRα) signaling have been tightly linked to cleft lip/palate in humans and mice, suggesting an evolutionarily conserved role in craniofacial development. During embryo development, the upper lip is formed by highly coordinated development of medial nasal process (MNP), lateral nasal process (LNP) and maxillary processes (MxP), all of which are originating from neural crest cells (NCCs). My previous study reveals that PDGFRα signaling is required to maintain MNP cell proliferation and regulate NCC migration, and indicates a role for small GTPase Rac1 in these processes downstream of PDGFRα. This proposed research is aimed at characterizing the role of Rac1 signaling in mediating PDGFRα regulation on MNP and NCC development. In aim one, I will use a newly generated unbiased Wnt1-Cre2 allele, to ablate Rac1 function specifically in NCCs, and characterize the mutant craniofacial phenotype at histological, cellular and molecular levels. The epistatic effect between Rac1 and PDGFRα will be tested by inactivating a single allele of each gene in NCCs. I will further rescue the craniofacial phenotype of PDGFRαfl/fl; Wnt1-Cre2 mice by driving expression a constitutively active form of Rac1 in NCCs in vivo. In aim two, I will examine how PDGFRα signaling regulates Rac1 activity during MNP morphogenesis. I will analyze the activity of immediate downstream signaling pathways in PDGFRα deficient MNP, and examine of the interaction between the identified signaling pathways and Rac1 activity. Aim three is designed to characterize the expression of PDGFRα transcriptional targets Cdc42ep3 (CEP3) and its role in craniofacial development. CEP3 will be examined with in vitro assays and gene-targeting method. Results of the proposed works will delineate the signaling cascade by which PDGFRα regulates NCC and MNP development, at the levels of signaling transduction and transcription. The results of proposed research will provide novel information to understand the fundamental mechanisms of MNP development and upper lip morphogenesis. This study will hold important benefit in treatment of cleft lip, to ultimately reduce the occurrence of this birth defect in humans.