The Role of SPECC1L cytoskeletal protein in craniofacial development and malformation

项目总结 口腔裂是美国最常见的出生缺陷之一，每800名活产儿中就有1名发生。这个 医疗、教育服务和生产力损失的终身成本平均超过100,000美元 受影响者。虽然已经确定了一些起作用的基因，但仍有必要继续 了解潜在的致病机制。我们在一部小说中发现了第一个从头开始的突变 斜面裂患者细胞骨架SPECC1L基因的研究SPECC1L突变的最新鉴定 在综合征性和非综合征性唇腭裂患者中，对细胞和 SPECC1L功能的分子机制将与严重面部和普通面部直接相关 畸形。我们的数据显示Spec1在胚胎8.5天的神经皱折中表达，包括在迁移前 脑神经脊细胞(CNCCs)。在后来的E9.5中，它在迁移后的CNCC中表达，在 颧弓位于E10.5。为了研究SPECC1L函数，我们生成了严重的、亚纯的和 Spec1缺乏症的条件性小鼠等位基因。严重的小鼠等位基因在E9.5岁时是胚胎致死的 移行后CNCC的神经管闭合缺陷和不完全分层。亚形的 等位基因表现为不完全穿透性围产儿脑外显型。中度突变体与一个 亚型和1个严重等位基因表现出高度渗透性的腭部闭合延迟。此外，SPECC1L缺陷 在体外培养的细胞中，细胞显示黏附连接(AJ)改变和PI3K-AKT信号减少 在体内，在Spec11突变组织中。细胞-细胞接触的调制不仅对CNCC分层很重要 从神经外胚层，但也为CNCC的“集体”迁移到其指定的目的地。中环 该项目的假设是，SPECC1L通过调节细胞黏附和集体迁移来调节细胞黏附和集体迁移 PI3K-AKT信号转导上皮细胞和间充质细胞与细胞接触的密度。在目标1中，我们建议 用我们的亚形、重度和非超微结构研究SPEC11剂量对颅面发育的影响。 有条件的小鼠等位基因。在目标2中，我们将研究SPECC1L对AJ稳定性和PI3K-AKT的调节作用 利用PI3K-AKT通路的小分子调节剂进行信号转导。我们还将探索这一机制 SPECC1L对AKT稳定性的潜在调节作用。在目标3中，我们将使用实时成像来定量评估 集体细胞行为的改变。分析将在体外培养的E8.5神经板外植体中进行， CNCC标有WNT1-CRE或SOX10-CRE。这些研究的成功完成将建立一种新的 面部形态发生过程中AJs、细胞极性和PI3K-AKT信号之间基于SPECC1L的联系 上颚闭合。了解SPECC1L剂量或功能缺陷与 集体细胞迁移将为未来治疗或预防口腔颌面部疾病提供靶点 裂开。

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.