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，全球每700名新生儿中约有1名患有唇裂。它是由环境或遗传因素破坏正常上唇发育引起的。这项研究的长期目标是了解上唇发育和口面裂发病机制。血小板衍生生长因子受体α（PDGFRα）信号转导的突变与人类和小鼠的唇腭裂密切相关，表明在颅面发育中具有进化保守的作用。在胚胎发育过程中，上唇由内鼻突（MNP）、外鼻突（LNP）和上颌突（MxP）高度协调发育而成，三者均起源于神经嵴细胞（NCCs）。我以前的研究表明，PDGFRα信号传导是维持MNP细胞增殖和调节NCC迁移所必需的，并表明小GTdR Rac 1在PDGFRα下游的这些过程中发挥作用。这项拟议的研究旨在表征Rac 1信号传导在介导PDGFRα调节MNP和NCC发展中的作用。在目标一，我将使用一个新产生的无偏Wnt 1-Cre 2等位基因，消融Rac 1功能，特别是在NCC，并在组织学，细胞和分子水平上的突变颅面表型的特点。将通过灭活NCC中每个基因的单个等位基因来检测Rac 1和PDGFRα之间的上位效应。我会进一步拯救颅面表型 PDGFRαfl/fl; Wnt 1-Cre 2小鼠的体内NCC中Rac 1组成型活性形式的表达。在目标二中，我将研究PDGFRα信号在MNP形态发生过程中如何调节Rac 1活性。我将分析PDGFRα缺陷型MNP中直接下游信号通路的活性，并检查所鉴定的信号通路与Rac 1活性之间的相互作用。目的三研究PDGFRα转录靶点Cdc 42 ep 3（CEP 3）在颅颌面发育中的表达及其作用。CEP 3将通过体外测定和基因靶向方法进行检查。本研究的结果将在信号转导和转录水平上描述PDGFRα调节NCC和MNP发育的信号级联反应。该研究结果将为理解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有重要意义，最终减少人类这种出生缺陷的发生。

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.