Role of cellular metabolism in palate morphogenesis

项目摘要 腭裂是最常见的先天性出生缺陷之一，全世界的患病率为1/700活产。 人类连锁研究表明，与胆固醇代谢相关的基因突变或异常 母亲的胆固醇饮食导致颅面畸形，如腭裂。然而，在很大程度上， 胆固醇分泌紊乱如何导致腭裂在我们的初步研究中，我们发现老鼠 胆固醇-C5-去饱和酶（Sc 5d）缺失，表现为腭裂，通过细胞减少， 在腭形成过程中增殖。Sonic Hedgehog（SHH）信号传导，对正常腭至关重要 形成，在Sc 5d突变小鼠中受损。初级纤毛，一种类似触角的结构， 在Sc 5d突变小鼠的腭间充质细胞中，质膜上的刺猬信号发生变形。 我们还发现，转录后蛋白质修饰和非编码RNA的表达发生了改变， Sc 5d突变小鼠的上颚。有趣的是，虽然胆固醇合成在缺乏 对于7-脱氢胆固醇还原酶（Dhcr 7）基因，这是关键的胆固醇合成后，SC 5D， 这些小鼠表现出腭裂，腭裂率低于10%。在此基础上，在本项目中， 将检验一种假设，即在Sc 5d突变小鼠中升高的胆固醇前体-- 在腭裂发病机制中的作用。我们有三个具体目标：（1）确定特定胆固醇 通过测试胆固醇中间体- Sc 5d突变小鼠，干扰hedgehog受体运动和初级纤毛形成;（2）鉴定 通过进行蛋白质组学研究， 使用Sc 5d突变体、Dhcr 7突变体和对照小鼠的腭部进行分析;（3）鉴定非编码RNA， 通过分析胆固醇代谢受损的调节基因， microRNA（短的非编码RNA）的作用机制及其对腭裂相关基因的调控 在Sc 5d突变小鼠中发生了特异性改变。在我们强有力的初步工作的基础上，我们期待这项研究 将系统地研究胆固醇代谢的作用（在细胞，代谢，蛋白质组学，和后， 转录调控水平）在小鼠腭裂，结果将导致创新的预防， 胆固醇相关颅面出生缺陷的诊断和治疗。

Project Abstract Cleft palate is one of the most common congenital birth defects, with a prevalence of 1/700 live births worldwide. Human linkage studies have shown that either genetic mutations related to cholesterol metabolism or abnormal maternal cholesterol diets lead to craniofacial deformities such as cleft palate. However, it is largely unknown how disturbances in cholesterol production result in cleft palate. In our preliminary studies, we found that mice with loss of sterol-C5-desaturase (Sc5d) displayed cleft palate with complete penetrance through decreased cell proliferation during palate formation. Sonic hedgehog (SHH) signaling, which is crucial for normal palate formation, was compromised in Sc5d mutant mice. The primary cilium, an antenna-like structure receiving hedgehog signals on the plasma membrane, was deformed in palatal mesenchymal cells of Sc5d mutant mice. We also found that posttranscriptional protein modification and expression of non-coding RNAs was altered in the palate of Sc5d mutant mice. Interestingly, while cholesterol synthesis is inhibited similarly in mice deficient for the 7-dehydrocholesterol reductase (Dhcr7) gene, which is crucial for cholesterol synthesis right after SC5D, these mice display cleft palate with a penetrance lower than 10%. Based on this foundation, in this project we will test the hypothesis that lathosterol, a cholesterol precursor that is elevated in Sc5d mutant mice, plays crucial roles in the pathogenesis of cleft palate. We have three specific aims; (1) To determine how a specific cholesterol intermediate interferes with SHH signaling by testing how lathosterol, a cholesterol intermediate accumulated in Sc5d mutant mice, interferes with hedgehog receptor movement and primary cilium formation; (2) To identify altered proteins and modifications in the developing palate of Sc5d mutant mice by conducting proteomic analyses using the palate of Sc5d mutant, Dhcr7 mutant, and control mice; (3) To identify non-coding RNAs and their regulated genes influenced by impaired cholesterol metabolism through analysis of the regulatory mechanism(s) of microRNAs (short non-coding RNAs) and their regulation of genes associated with cleft palate that are specifically altered in Sc5d mutant mice. Building on our strong preliminary work, we expect this study will systematically investigate the roles of cholesterol metabolism (at the cellular, metabolic, proteomic, and post- transcriptional regulation levels) in cleft palate in mice, and the results will lead to innovations in the prevention, diagnosis, and treatment of cholesterol-related craniofacial birth defects.