The Role of SPECC1L cytoskeletal protein in craniofacial development and malformation

项目摘要 口面裂是美国最常见的出生缺陷之一，发生在1/800活产婴儿中。的 医疗、教育服务和生产力损失的终身成本平均超过10万美元， 受影响的人。虽然已经确定了一些贡献基因，但仍然需要 了解潜在的发病机制。我们在一个新的基因组中 面斜裂患者细胞骨架SPECC1L基因的研究SPECC1L突变的最新鉴定 综合征型和非综合征型唇腭裂患者的研究表明， SPECC1L功能的分子机制将与严重和常见的面部疾病直接相关， 畸形我们的数据显示Specc1l在E8.5的神经褶皱中表达，包括在迁移前的神经褶皱中。 颅神经嵴细胞（CNCC）。随后在E9.5，它在迁移后CNCC中表达，并且在 鳃弓在E10.5。为了研究SPECC1L函数，我们生成了严重的，亚纯的， Specc1l缺陷的条件性小鼠等位基因。重度小鼠等位基因在E9.5时具有胚胎致死性， 有缺陷的神经管（NT）闭合和迁移后CNCC的不完全分层。亚纯的 等位基因表现为不完全外显的围产期露脑表型。中度突变体， 亚型和一个严重等位基因显示高度渗透性腭闭合延迟。此外，SPECC1L缺陷型 细胞显示改变的粘附连接（AJs）和减少的PI3K-AKT信号传导，在体外培养的细胞和 在Specc11突变体组织中体内。细胞-细胞接触的调节不仅对于CNCC分层是重要的， 从神经外胚层，但也用于CNCC的"集体"迁移到其定义的目的地。中央 该项目的假设是SPECC1L通过调节细胞粘附和集体迁移来调节细胞粘附和集体迁移。 通过PI3 K-AKT信号传导的上皮和间充质细胞-细胞接触密度。在目标1中，我们建议 研究Specc1l剂量对颅面发育的影响， 条件小鼠等位基因。在目标2中，我们将研究SPECC1L介导的AJ稳定性和PI3K-AKT 使用PI3K-AKT途径的小分子调节剂进行信号传导。我们还将探讨机制 潜在的SPECC1L介导AKT稳定性。在目标3中，我们将使用实时成像来定量评估 集体细胞行为的变化。将在离体培养的E8.5神经板外植体中进行分析， 用Wnt1-Cre或Sox10-Cre标记的CNCC。成功完成这些研究将建立新的 基于SPECC1L的面部形态发生过程中AJs、细胞极性和PI3K-AKT信号传导之间的联系， 腭闭合了解SPECC1L剂量或功能缺陷与 集体细胞迁移将为未来针对口面的治疗或预防策略提供目标 裂缝

PROJECT SUMMARY Orofacial clefts are among the most common birth defects in the U.S., occurring in 1/800 live-births. The lifetime cost for medical treatment, educational services and lost productivity averages more than $100,000 per affected person. While a number of contributory genes have been identified, there is a continued need to understand the underlying pathogenetic mechanisms. We identified the first de novo mutations in a novel cytoskeletal SPECC1L gene in patients with oblique facial clefts. Recent identification of SPECC1L mutations in patients with syndromic and nonsyndromic cleft lip and palate suggests that insights into the cellular and molecular mechanism of SPECC1L function will be directly relevant to both severe and common facial malformations. Our data show that Specc1l is expressed in the neural folds at E8.5, including in pre-migratory cranial neural crest cells (CNCCs). Later at E9.5, it is expressed in post-migratory CNCCs, and in the branchial arches at E10.5. To study SPECC1L function, we have generated severe, hypomorphic and conditional mouse alleles of Specc1l deficiency. The severe mouse allele is embryonic lethal at E9.5 with defective neural tube (NT) closure and incomplete delamination of post-migratory CNCCs. The hypomorphic allele shows incompletely penetrant perinatal exencephaly phenotype. Moderate mutants with one hypomorphic and one severe allele show highly penetrant palate closure delay. In addition, SPECC1L-deficient cells show altered adherens junctions (AJs) and reduced PI3K-AKT signaling, both in vitro in cultured cells and in vivo in Specc1l mutant tissue. Modulation of cell-cell contacts is important not only for CNCC delamination from the neuroectoderm, but also for “collective” migration of CNCCs to their defined destinations. The central hypothesis of this project is that SPECC1L modulates cell adhesion and collective migration by regulating the density of epithelial and mesenchymal cell-cell contacts through PI3K-AKT signaling. In Aim 1, we propose to investigate the effect of Specc1l dosage on craniofacial development using our hypomorphic, severe, and conditional mouse alleles. In Aim 2, we will investigate SPECC1L mediation of AJ stability and PI3K-AKT signaling using small molecule modulators of PI3K-AKT pathway. We will also explore the mechanism underlying SPECC1L mediation of AKT stability. In Aim 3, we will use live-imaging to quantitatively assess changes in collective cell behavior. Analyses will be conducted in ex vivo cultured E8.5 neural plate explants, with CNCCs marked with Wnt1-Cre or Sox10-Cre. Successful completion of these studies will establish novel SPECC1L-based links between AJs, cell polarity and PI3K-AKT signaling during facial morphogenesis and palate closure. Understanding the correlation between SPECC1L dosage or functional deficiency and collective cell migration will provide targets for future therapeutic or preventative strategies against orofacial clefting.