BACE2通过Wnt信号参与神经嵴诱导影响颅面发育的分子机制研究

Craniofacial abnormities in most case caused by developmental failure of cranial cartilage and bones are most common congenital born deficiencies. These deficiencies are reasoned by abnormal genesis of anterior neural crest cells. Although, parts of these abnormities can be rectified via surgical operations, some severe cases still lack effective way to cure. To better understand the etiology of congenital cranial defects and thereby establishing prenatal screening in early pregnant period with sensitive genes, much work should be done to explore the mechanisms of cranial neural crest development and relative gene-regulatory-networks. In this program, we use neural crest EGFP transgenic Xenopus and Cas-9 mediated BACE2-knock out Xenopus as model system, our preliminary data imply that inhibition the β-site APP cleaving enzyme2 BACE2 may block cranial cartilage development and therefore, result in craniofacial defects in a Wnt signaling dependent manner. To convince our hypothesis and clarify BACE2 functions in cranial cartilage development during both physical and pathological conditions, we plan to search for the time window that BACE2 involves in during cranial neural crest development and screen its proteolytic substrate(s) from candidate genes which participate in Wnt signaling transduction. Thereafter, we can provide theoretical supports for BACE2 as a sensitive gene for cranial abnormities screen and drug design in the future.

颅面畸形是一类常见出生缺陷，相当一部分是由神经嵴细胞异常发育引起颅面软骨和骨的形成缺陷所致。临床上主要通过手术矫正，一些严重病例尚缺乏有效治疗手段。了解颅面神经嵴发育机制以及相关基因作用方式对于更加深入认识颅面畸形病因学以及选择合适易感基因建立孕早期产前筛查项目降低先天缺陷率具有积极现实的意义。本项目以模式动物爪蛙作为研究系统，构建了颅面神经嵴转基因品系和Cas-9介导的BACE2基因敲除爪蛙品系。前期工作提示阻断β-分泌酶2(BACE2)可能通过干扰Wnt信号影响颅面软骨形成，造成颅面缺陷。为验证这一全新假说，本项目拟探讨颅面神经嵴发育中BACE2作用时间，筛选并鉴定BACE2在Wnt信号中分子靶点，阐明其参与颅面软骨发育作用方式，揭示病理情况下引起颅面神经嵴发育异常造成颅面缺陷的分子机制，为其作为候选基因建立颅面缺陷产前筛查以及后续药物研发提供理论依据。

非洲爪蛙模式动物中阻断beta分泌酶2(BACE2)功能，胚胎表现出神经嵴发育缺陷表型。基于此我们进一步在细胞和小鼠模型中研究BACE2的功能及作用机制。在小鼠中我们敲除BACE2基因后，出生小鼠并未表现出明显的颅面异常，但是胚胎表现出体型变小，体重增长对高脂饮食抵抗等表型。生化水平上在U251细胞中，BACE2和蛋白合成延伸因子eEF2相互作用，eEF2在细胞内具有全长和剪切形式，这种剪切受到diphthamide修饰的调节。通过KD阻断DPH1基因，阻断diphthamide修饰后，爪蛙胚胎缺乏diphthamide修饰，eEF2剪切形式变少，神经嵴发育缺陷；在小鼠DPH1基因功能失活的突变胚胎中（E14），也观察到唇腭裂表型。我们的工作可能揭示了diphthamide修饰调节由BACE2参与的eEF2剪切影响神经嵴发育的新的分子机制.

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