Role of SPECC1L cytoskeletal protein in palate elevation dynamics

项目摘要 口面裂涉及唇和腭是最常见的先天性颅面畸形发生 作为>400种综合征的一部分或作为约1/700活产的孤立表型。非综合征性腭裂仅由 本身影响1/1700的儿童，女性发病率增加2：1。一生的医疗费用， 教育服务和生产力损失平均超过10万美元。胚胎 腭发生涉及从上颌骨水平隆起并融合的两侧垂直生长的搁板 舌头上面。虽然腭发育已经研究了世纪，但尚不清楚腭发育是如何发生的， 在提升过程中，搁板会从垂直方向重新定向为水平方向。造成这种知识差距的一个原因是 这个过程非常迅速，因此很难计时和捕捉。另一个原因是缺乏评估方法 腭部抬高和具有良好表征的腭部抬高延迟的小鼠模型。我们的研究表明， 腭架在子宫内不到3小时内升高，并且有一个确定的胚胎时间窗， 提升我们还评估了细胞增殖、细胞定向和肌动球蛋白的动态相互作用 在正常的腭架抬高的基础上的收缩。我们已经建立了新的方法来使用MRI 用于子宫内成像，并使用有限元分析来模拟腭架抬高。此外，我们一直在 研究细胞骨架蛋白SPECC 1 L在腭发育中的作用。我们发现了第一个新生的 口面裂患者的常染色体显性SPECC 1 L突变。我们和其他人已经证明， SPECC 1 L突变的患者聚集在第二螺旋艾德螺旋结构域（CCD 2）或钙调蛋白同源性中 先天性心脏病（CHD）通常表现为距离过远，腭裂和脐膨出等表型。使用 多个Specc 1 l小鼠等位基因，我们已经确定了SPECC 1 L的缺失导致腭架延迟 提升有趣的是，小鼠中的框内CCD 2特异性突变（缺失，点突变）导致更多的细胞凋亡。 严重的腭架抬高延迟，表明功能获得。在细胞水平上，CD 2CCD 2导致 SPECC 1 L沿着的核周错误定位，伴随着细胞质丝状肌动蛋白和非肌肉肌动蛋白的破坏， 肌球蛋白Ⅱ最后，我们的初步数据表明，在哺乳动物CCD 2突变体中， 补充酸。在目标1中，我们将研究正常和异常的细胞和组织力学 使用离体和体内磁共振成像和计算的CCD 2等位基因的腭部升高 建模在目标2中，我们将研究功能获得的细胞和分子机制， 使用最先进的蛋白质组学分析的CCD 2突变细胞。在目标3中，我们将确定母体叶酸 补充剂挽救了CCD 2突变体中的腭部抬高缺陷。成功完成这些研究将 对正常和异常腭架期间的组织动力学和细胞信号传导提供重要见解 提高，以及产妇叶酸补充剂的作用，这将提供未来的目标， 口面裂的翻译策略。

PROJECT SUMMARY Orofacial clefts involving the lip and palate are the most common congenital craniofacial malformation that occur as part of >400 syndromes or as an isolated phenotype in ~1/700 live-births. Non-syndromic cleft palate only by itself afflicts 1/1700 children with a 2:1 increased incidence in females. The lifetime cost for medical treatment, educational services, and lost productivity averages more than $100,000 per affected person. Embryonic palatogenesis involves bilateral vertical outgrowth of shelves from the maxilla that elevate horizontally and fuse above the tongue. While palatogenesis has been studied for more than a century, it is not clear how palatal shelves reorient from the vertical to horizontal direction during elevation. One reason for this knowledge gap is that this process is rapid and therefore hard to time and capture. Another is a lack of methodologies to assess palate elevation and of mouse models with a well-characterized palate elevation delay. Our studies show that palatal shelves elevate in less than 3 hours in utero and that there is a defined embryonic window of time for elevation. We have also assessed the dynamic interplay of cell proliferation, cell orientation and actomyosin contraction that underlies normal palatal shelf elevation. We have established novel methodologies to use MRI for in utero imaging and to use finite element analysis to model palatal shelf elevation. In addition, we have been studying the role of cytoskeletal scaffolding protein SPECC1L in palatogenesis. We identified the first de novo autosomal dominant SPECC1L mutations in patients with orofacial clefts. We and others have now shown that patients with SPECC1L mutations clustered in the second coil ed coil domain (CCD2) or calponin homology domain (CHD) commonly manifest hypertelorism, cleft palate and omphalocele among other phenotypes. Using multiple Specc1l mouse alleles, we have established that loss of SPECC1L leads to a delay in palatal shelf elevation. Interestingly, in-frame CCD2 specific mutations (deletions, point mutations) in mice result in a more severe palatal shelf elevation delay, indicating a gain-of-function. At the cellular level, CCD2 leads to perinuclear mislocalization of SPECC1L along with a disruption of cytoplasmic filamentous actin and non-muscle myosin II. Lastly, our preliminary data show that cleft palate in CCD2 mutants is rescued upon maternal folic acid supplementation. In Aim 1, we will study the cell and tissue mechanics underlying both normal and abnormal palate elevation in CCD2 alleles using ex vivo and in vivo magnetic resonance imaging and computational modeling. In Aim 2, we will investigate the cellular and molecular mechanisms underlying the gain-of-function in CCD2 mutant cells using state-of-the-art proteomic analyses. In Aim 3, we will determine how maternal folate supplementation rescues palate elevation defects in CCD2 mutants. Successful completion of these studies will provide critical insights into tissue dynamics and cell signaling during normal and abnormal palatal shelf elevation, as well as into the effect of maternal folic acid supplementation, which will provide targets for future translational strategies against orofacial clefting.