Role of Cytoskeletal Protein SPECC1L in Facial Morphogenesis and Facial Clefting

项目总结(见说明)： 口腔裂是美国最常见的出生缺陷之一，每750名活产儿中就有1名发生。每个受影响的人一生的医疗、教育服务和丧失生产力的成本平均超过10万美元。虽然大多数口腔裂隙会导致唇裂合并或不合并腭裂(CL/P)，但也有一小部分会导致从口腔延伸到眼睛的斜面裂隙(ObFC)。 尽管不太常见，但对ObFC的细胞机制--由Paul Tessier于1976年首次明确分类--的见解仍然难以捉摸。我们已经确定了两个新发生的SPECC1L突变在ObFC患者中。我们对斑马鱼和苍蝇的研究为了解SPECC1L的功能提供了重要的见解，到目前为止，还没有科学出版物对其进行研究。敲除一个以前没有特征的斑马鱼SPECC1L同源物扰乱了脑神经脊线(NC)，并导致了戏剧性的 面部结构的丧失，从而将SPECC1L在面部形态发生中的功能扩展到其他脊椎动物。此外，敲除唯一没有特征性的果蝇直系同源表型-在很大程度上-整合素信号通路中已知的苍蝇突变体-表现出细胞黏附和迁移缺陷。 此外，我们的细胞和分子分析表明，SPECC1L是一种新的细胞骨架交联蛋白，它同时与微管和肌动蛋白细胞骨架相互作用。SPECC1LGFP的瞬时表达稳定了微管的一部分，而SPECC1L的敲除导致了肌动蛋白细胞骨架的缺陷重组，并损害了细胞的黏附和迁移。再加上老鼠在发育中的面部隆起中的速度I表达，这些结果开始解释人类ObFC是如何在SPECC1L缺乏后发生的。这项建议的目的是建立一种小鼠模型，以测试哺乳动物面部形态发生中SPECC1L缺陷的发病机制(目标1)，并精确确定SPECC1L在细胞迁移和面部结构规范中的细胞(目标2)和分子(目标3)角色。

PROJECT SUMMARY (See instructions): Orofacial clefts are one of the most common birth defects in the U.S., occurring in 1/750 live births. The lifetime cost for medical treatment, educational services and lost productivity averages more than $100,000 per affected person. While the majority of orofacial clefts result in cleft lip with or without cleft palate (CL/P), a small percentage results in oblique facial clefts (ObFC) that extend from the oral cavity to the eye. Although less common, insights into the cellular mechanism of ObFC - first definitively classified by Paul Tessier in 1976 - have remained elusive. We have identified two de novo occurrences of SPECC1L mutations in patients with ObFC. Our studies in zebrafish and fly provide significant insight into SPECC1L function, which thus far had remained unstudied with no scientific publications. Knockdown of a previously uncharacterized zebrafish SPECC1L homolog perturbs cranial neural crest (CNC) and results in a dramafic loss of facial structures, thus extending SPECC1L function in facial morphogenesis to other vertebrates. In addition, knockdown of the sole uncharacterized Drosophila ortholog phenocopies - to an extraordinary extent - known fly mutants in the integrin-signaling pathway that exhibit cell adhesion and migration defects. Furthermore, our cellular and molecular analyses show that SPECC1L is a novel cytoskeletal cross-linking protein that interacts with both the microtubule and actin cytoskeletons. Transient expression of SPECC1LGFP stabilizes a subset of microtubules, while SPECC1L knockdown causes defective actin cytoskeleton reorganization and impairs cell adhesion and migration. Together with mouse Speed I expression in the developing facial prominences, these results begin to explain how human ObFC can arise following SPECC1L deficiency. The aim of this proposal is to develop a mouse model to test the pathogenetic mechanism of SPECC1L deficiency in mammalian facial morphogenesis (Aim 1) and to precisely define the cellular (Aim 2) and molecular (Aim 3) role of SPECC1L in CNC cell migration and specification of facial structures.