YAP/TAZ复合体在下颌骨发育中的功能及作用机制

Mandibular malformation is a quite common genetic disease, which is caused by defective mandibular development. Mandibula originates from the neural crest. The stages of mandibular development include the neural crest-derived mesenchymal cell migration, differentiation from mesenchymal to cartilage cells, morphogenesis, and bone formation etc. Proper regulation and coordination of these stages is pivotal for proper mandibular development. YAP/TAZ, core factors of Hippo signaling pathway, have been reported to regulate osteoblast and osteoclast differentiation as well as maintenance of bone homeostasis. However, due to early embryonic lethal of general knockout and neural crest-specific knockout mice, no report is currently available on the roles of YAP/TAZ specifically in mandibular development. In this project, we will establish mandibular arch-specific knockout mice using the dHand promoter, in combination with the zebrafish model system, to study the specific functions and mechanisms of YAP/TAZ in mandibular development based on the following three aims. 1) Phenotype studies of the mandibular arch-specific knockout mice. 2) Molecular mechanisms of YAP/TAZ in the mandibular development. 3) Roles of Yap/Taz in the mandibular development in zebrafish.

下颌骨畸形是常见的遗传疾病，由下颌骨的发育异常导致。下颌骨起源于神经嵴细胞，其发育经历了神经嵴来源的间充质细胞迁移、间充质细胞向软骨细胞分化、形态建成、成骨分化等过程，每个过程的精细调控和协调是下颌骨正常发育的关键。Hippo信号通路的核心作用因子YAP/TAZ可调控成骨分化、破骨分化及骨代谢稳态的维持，但由于YAP/TAZ的常规基因敲除和神经嵴细胞特异性敲除均早期致死，无法很特异地研究YAP/TAZ在下颌骨发育中的功能。针对这个问题，本项目将利用dHand启动子建立下颌骨前体组织下颌弓特异性基因敲除工具小鼠，并结合斑马鱼模式动物，致力于从以下三个部分研究YAP/TAZ在下颌骨发育中的功能及其作用机制：1）研究YAP/TAZ在下颌弓特异性敲除及过表达后的下颌发育表型；2）研究YAP/TAZ调控下颌骨发育的主要分子机制；3）小鼠中发现的表型和分子机制在斑马鱼中的验证。

本课题以dHand-Cre转基因小鼠作为模式动物，通过基因条件性敲除和过表达，运用分子生物学、细胞生物学和免疫学等手段，研究YAP/TAZ在下颌骨不同发育时期的表型，探讨其分子调控机制，阐明YAP/TAZ在下颌骨发育畸形干预治疗及下颌骨再生研究的作用。研究发现，YAP在E12.5和E15.5时期小鼠胚胎的下颌弓中均有表达；条件性敲除YAP后，在出生后发育阶段可抑制下颌骨髁突本身的发育以及髁突的软骨内骨化功能；通过生信分析YAP沉默后的颅神经嵴细胞O9-1，筛选出MAPK与Wnt通路作为颅颌面发育的重要信号通路。分离提取了髁突软骨干细胞（FCSC）后，慢病毒转染shRNA沉默和过表达了FCSC中YAP的表达，YAP可负向调控FCSC的凋亡率与成软骨分化能力，可正向调控FCSC的增殖活力。进一步研究发现沉默YAP后Wnt/βcatenin通路被激活，YAP可通过Wnt/β-catenin通路调控FCSC成软骨分化能力。证实了YAP/TAZ在下颌骨发育中的功能及其作用机制，为下颌骨发育畸形等疾病的病理机制、早期干预治疗及下颌骨缺损修复甚至再生提供新的思路及证据。