The Role of SPECC1L cytoskeletal protein in craniofacial development and malformation

SPECC1L细胞骨架蛋白在颅面发育和畸形中的作用

• 批准号: 9304185
• 负责人: Irfan Saadi
• 金额: $ 32.51万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304185
• 关键词: Adherens Junction; Adhesions; Affect; Alleles; Attenuated; Bilateral; Biological Assay; Branchial arch structure; Cell Adhesion; Cell Communication; Cell Density; Cell Polarity; Cell membrane; Cell physiology; Cells; Cephalic; Chemicals; Collaborations; Complex; Congenital Abnormality; Craniofacial Abnormalities; Cultured Cells; Cytoskeletal Proteins; Data; Defect; Destinations; Disease; Embryo; Embryonic Development; Epithelial; Exencephalies; Exhibits; Eye; Face; Family; Future; Genes; Genetic; Goals; Human; Image; In Vitro; Intercellular Junctions; Iowa; Japan; Knowledge; Link; Live Birth; Measures; Mediating; Mediation; Medical; Membrane; Mesenchymal; Modeling; Molecular; Morphogenesis; Movement; Mus; Mutate; Mutation; Neural Crest; Neural Crest Cell; Neural Fold; Neural Tube Closure; Neural tube; Neuroectoderm; Neuroepithelial; Neuroepithelial Cells; Operative Surgical Procedures; Opitz syndrome; Oral; Oral cavity; Orbital separation excessive; Outcome; Palate; Pathogenesis; Pathway interactions; Patients; Perinatal; Persons; Phenotype; Play; Prevention strategy; Productivity; Proto-Oncogene Proteins c-akt; Role; Sampling; Services; Severities; Signal Transduction; Stream; Syndrome; Teebi syndrome; Testing; Therapeutic; Tissues; Transmission Electron Microscopy; Ubiquitination; base; cell behavior; cell motility; cleft lip and palate; craniofacial; craniofacial development; density; dosage; improved; in vivo; inhibitor/antagonist; insight; life time cost; malformation; migration; mouse model; mutant; neural plate; new therapeutic target; novel; orofacial cleft; palatal shelves; palatogenesis; postnatal; small molecule

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.

项目概要 口面部裂是美国最常见的出生缺陷之一，发生在 1/800 的活产儿中。这 医疗、教育服务和生产力损失的终生成本平均每人超过 100,000 美元 受影响的人。虽然已经鉴定了许多贡献基因，但仍需要继续 了解潜在的发病机制。我们在小说中发现了第一个从头突变 斜面裂患者的细胞骨架 SPECC1L 基因。 SPECC1L 突变的最新鉴定 在患有综合征性和非综合征性唇腭裂的患者中，表明对细胞和 SPECC1L功能的分子机制将与严重和普通面部直接相关 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 颅神经嵴细胞（CNCC）。后来在 E9.5 中，它以迁移后 CNCC 表示，并在 鳃弓位于 E10.5。为了研究 SPECC1L 功能，我们生成了严重的、低效的和 Specc1l 缺陷的条件性小鼠等位基因。严重的小鼠等位基因在 E9.5 时是胚胎致死的 神经管 (NT) 闭合缺陷和迁移后 CNCC 不完全分层。亚构型 等位基因显示不完全渗透的围产期无脑畸形表型。中度突变体有一个 低等位基因和一个严重的等位基因表现出高度渗透性的腭闭合延迟。此外，SPECC1L 缺陷 在体外培养的细胞和 体内 Specc1l 突变组织中。细胞与细胞接触的调节不仅对于 CNCC 分层很重要 从神经外胚层，也可以将 CNCC“集体”迁移到其指定的目的地。中央 该项目的假设是 SPECC1L 通过调节细胞粘附和集体迁移 通过 PI3K-AKT 信号传导上皮细胞和间充质细胞-细胞接触的密度。在目标 1 中，我们建议 使用我们的低等位、严重和 条件小鼠等位基因。在目标 2 中，我们将研究 SPECC1L 对 AJ 稳定性和 PI3K-AKT 的调节 使用 PI3K-AKT 通路小分子调节剂进行信号传导。我们还将探索机制 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。分析将在离体培养的 E8.5 神经板外植体中进行， CNCC 标有 Wnt1-Cre 或 Sox10-Cre。成功完成这些研究将建立新的 面部形态发生过程中 AJ、细胞极性和 PI3K-AKT 信号之间基于 SPECC1L 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口面部的治疗或预防策略提供目标 裂开。