Role of Cytoskeletal Protein SPECC1L in Facial Morphogenesis and Facial Clefting

项目总结（见说明）： 口面裂是美国最常见的出生缺陷之一，发生在1/750活产婴儿中。每个受影响者的医疗、教育服务和生产力损失的终身费用平均超过100 000美元。虽然大多数口面裂导致唇裂伴或不伴腭裂（CL/P），但一小部分导致从口腔延伸到眼睛的斜面裂（ObFC）。 虽然不太常见，但对ObFC细胞机制的见解-首先由Paul Tessier在1976年明确分类-仍然难以捉摸。我们已经确定了两个从头发生的SPECC 1 L突变的ObFC患者。我们对斑马鱼和苍蝇的研究为SPECC 1 L功能提供了重要见解，迄今为止该功能尚未研究，也没有科学出版物。敲除先前未表征的斑马鱼SPECC 1 L同源物扰乱了颅神经嵴（CNC），并导致了一个戏剧性的 面部结构的丧失，从而将SPECC 1 L在面部形态发生中的功能扩展到其他脊椎动物。此外，敲除唯一的未经鉴定的果蝇直系同源物表型-在非常大的程度上-已知的苍蝇突变体在整合素信号通路中表现出细胞粘附和迁移缺陷。 此外，我们的细胞和分子分析表明，SPECC 1 L是一种新型的细胞骨架交联蛋白，与微管和肌动蛋白细胞骨架相互作用。SPECC 1 LGFP的瞬时表达稳定了微管的一个子集，而SPECC 1 L敲低导致有缺陷的肌动蛋白细胞骨架重组，并损害细胞粘附和迁移。连同小鼠的速度I表达在发展中的面部皱纹，这些结果开始解释如何人类ObFC可以出现以下SPECC 1 L缺陷。该提案的目的是开发一种小鼠模型，以测试哺乳动物面部形态发生中SPECC 1 L缺陷的发病机制（目标1），并精确定义SPECC 1 L在CNC细胞迁移和面部结构特化中的细胞（目标2）和分子（目标3）作用。

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.