Characterizing the mechanisms of PDGFRalpha regulation in upper lip development

描述(申请人提供)：唇裂是最常见的出生缺陷之一。唇裂是由正常的颅面发育障碍引起的，全世界大约每500名新生儿中就有1人患有唇裂。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(PDGFR)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。在胚胎发育过程中，上唇由内侧鼻突和上颌突融合而成，两者都起源于神经脊细胞。本研究旨在研究PDGFR在MNP发育和上唇形态发生过程中神经脊细胞的作用。首先，我们将建立一个新的转基因株系Alx3Cre来研究PDGFR在MNP发育中的作用。用免疫组织化学方法检测条件基因敲除胚胎和对照胚胎的MNP中的细胞增殖和细胞存活。划痕实验和Transwell实验将分析PDGFR信号在MNP细胞迁移中的作用。在第二个特定目标中，我们将研究PDGFR在神经脊细胞中的特定作用。在条件基因敲除胚胎和对照胚胎中，将谱系追踪法和体外外植体培养方法与免疫组织化学实验相结合。在第三个特定目标中，我们将利用PDGFR信号突变体PDGFR？PI3K/PI3K小鼠，分析参与PI3K信号的PDGFR在MNP和神经脊发育中的作用。第一个和第二个特定目标中提出的体外细胞迁移分析和体内小鼠遗传学研究将在PDGFR？PI3K/PI3K和对照胚胎上进行。建议的工作结果将揭示PDGFR在MNP和上唇形态发生过程中神经脊细胞发育中的基础作用。研究结果将为理解MNP和上唇形成的基本机制提供新的信息。本研究将有助于今后唇裂的治疗和预防，最终减少这一出生缺陷在新生儿中的发生。

DESCRIPTION (provided by applicant): Cleft lip is one of most common birth defects. Caused by disruption of normal craniofacial development, cleft lip affects approximately 1 in 500 newborns worldwide. 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 ¿ (PDGFR¿) 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 fusion of medial nasal process (MNP) and maxillary processes, both originating from neural crest cells. This proposed research is aimed at characterizing the role of PDGFR¿ in development of MNP and of neural crest cells during upper lip morphogenesis. First, we will generate a novel transgenic line Alx3Cre to examine the role of PDGFR¿ specifically in MNP development. Cell proliferation and cell survival will be assayed in the MNP of conditional knockout and control embryos using immunohistochemistry methods. Scratch assay and transwell assay will be carried out to analyze the role of PDGFR¿ signaling in MNP cell migration. In the second specific aim, we will examine the role of PDGFR¿ specifically in neural crest cells. Lineage tracing method and in vitro explants culture approaches will be combined with immunohistochemistry experiments in the conditional knockout and control embryos. In the third specific aim, we will analyze the role of PDGFR¿ engaged PI3K signaling in MNP and neural crest development using PDGFR¿ signaling mutant PDGFR¿ PI3K/PI3K mice. In vitro cell migration assays and in vivo mouse genetic studies proposed in first and second specific aims will be carried out on PDGFR¿ PI3K/PI3K and control embryos. Results of the proposed works will reveal the fundamental role of PDGFR¿ in MNP and neural crest cell development during upper lip morphogenesis. The results of proposed research will provide novel information to understand the fundamental mechanisms of MNP and upper lip formation. This study will benefit treatment and prevention of cleft lip in the future, to ultimately reduce the occurrence of this birth defect in newborns.