Role of SPECC1L cytoskeletal protein in palate elevation dynamics

项目总结 唇腭裂是最常见的先天性颅面畸形。 作为&gt；400综合征的一部分或作为~1/700活产婴儿的孤立表型。非综合征性腭裂仅限于 其本身影响1/1700名儿童，女性发病率增加2：1。医疗的终生成本， 教育服务，生产力损失平均每个受影响的人超过100,000美元。胚胎 腭裂发生于上颌骨两侧垂直延伸的搁板上，水平升高并融合在一起。 在舌头上方。虽然对腭裂的研究已有一个多世纪的历史，但目前尚不清楚 工具架在立面过程中从垂直方向重定向为水平方向。造成这种知识鸿沟的一个原因是 这个过程很快，因此很难计时和捕捉。另一个问题是缺乏评估方法。 腭部抬高和具有良好特征的腭部抬高延迟的小鼠模型。我们的研究表明 腭架在宫内不到3小时内升高，并且有一个明确的胚胎时间窗 立面。我们还评估了细胞增殖、细胞定位和肌动球蛋白之间的动态相互作用。 在正常的腭架抬高基础上的收缩。我们已经建立了使用核磁共振的新方法 用于宫内成像，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SPECC1L在腭裂发生中的作用。我们发现了第一个从头开始的 口裂患者常染色体显性遗传性SPECC1L突变的研究我们和其他人现在已经证明 SPECC1L突变患者聚集在第二螺旋线圈结构域(CCD2)或钙蛋白同源区域 领域(CHD)通常表现为染色体增多症、腭裂和脐膨出等表型。vbl.使用 多个SPEC1L小鼠等位基因，我们已经证实SPECC1L的丢失会导致腭架的延迟 立面。有趣的是，小鼠的框内CCD2特异性突变(缺失、点突变)会导致更多的 严重的腭架抬高延迟，表明获得了功能。在细胞水平上，CCD2导致 SPECC1L核周定位错误并伴有细胞质丝状肌动蛋白和非肌肉的破坏 肌球蛋白II。最后，我们的初步数据显示，ccd2突变体中的腭裂在母体叶酸的作用下得以挽救。 补充酸性物质。在目标1中，我们将研究正常和异常背后的细胞和组织力学 用体外和体内磁共振成像和计算方法研究CCD2等位基因的腭部升高 模特儿。在目标2中，我们将研究潜在的细胞和分子机制的功能获得 使用最先进的蛋白质组学分析CCD2突变细胞。在目标3中，我们将确定母体叶酸如何 补充剂挽救了CCD2突变体中的腭部抬高缺陷。成功完成这些研究将 提供对正常和异常腭裂过程中的组织动力学和细胞信号的重要见解 提高，以及母亲补充叶酸的效果，这将为未来提供目标 防止口裂的翻译策略。

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.