利用维甲酸诱导及Wnt5a敲除两种腭裂模型研究舌发育的分子调控机制及其对腭裂发生的影响

The effect of tongue developmental abnormality to palatal shelves' horizontal elevation, contact and fusion becomes to be a new hot spot of mechanism studies of cleft palate formation recently. Nevertheless, the common and collaborative molecular mechanisms in regulation both of tongue and palate development have not been explored. In our current research, the similar phenotype of cleft palate with tongue abnormality was found in retinoic acid (RA)-induced mouse and Wnt5a gene knock-out models respectively, and the expression of myogenic regulatory factors (MRFs) was down- regulated. The gene microarray demonstrated the different expression of Wnt signaling pathway between wild type and RA- induced cleft palate, suggesting RA and Wnt5a may regulate the development of tongue and palate. On this basis, the project will firstly determine the spatial-temporal dynamic changes of tongue development and the mechanism of tongue deformation in above two mouse models, combining with myogenic- inducing differentiation of myoblast cell lines in vitro, to investigate the mechanisms of RA and Wnt5a on regulating the differentiation of lingual skeleton muscles. Furthermore, the genomic and proteomic analysis will be performed on the tongue and palate at the different stages of their development in the above animal models, and the common and collaborative molecular networks of the regulation of tongue and palate development will be investigated, then the network of target proteins will be elaborated, and interactive networks of target molecules will be illustrated during the cleft palate and tongue abnormality. Finally, under the utilization of 3-D microgravity organ culture system, the direct relationship between abnormal tongue formation and cleft palate will be explored, and the direct effect of target genes and proteins on the development of palate and tongue will be validated. This research provides a further realize to the mechanisms of tongue development, and a theoretical basis and experimental foundation for the prevention and treatment of clinical cleft palate with tongue abnormality.

舌发育异常导致的腭上抬及接触融合障碍是先天性腭裂发病机制研究的新热点，但调控舌发育的分子机制却知之甚少。我们在维甲酸诱导和Wnt5a敲除两种腭裂模型中发现相似的舌异常表型、舌肌中的肌向分化标记物MRF家族表达下调以及Wnt信号通路的基因差异表达，提示了维甲酸和Wnt5a可能调控舌的发育。本项目拟首先在体内利用上述模型确定舌发育时空动态变化并探究舌发育异常的机制，在体外肌向诱导的成肌细胞系中进一步加以验证；其次通过基因和蛋白组学研究获得上述模型不同时相的舌腭发育的调控分子网络，加以验证并绘出腭裂发生与舌发育异常的靶蛋白相互作用网络图；最后利用模拟体内舌腭颌的三维微重力器官培养体系，验证靶基因和靶蛋白对舌腭发育的直接调控作用，探讨特定环境（维甲酸）与基因（Wnt5a信号分子）交互作用下腭裂发生与舌发育异常的关联。本课题有望加深了解舌发育的分子机理，并为舌腭发育异常的防治提供可能的科学依据。

舌发育异常导致的腭上抬及接触融合障碍是先天性腭裂发病机制研究的新热点，但调控舌发育的分子机制却知之甚少。我们在维甲酸诱导和 Wnt5a 敲除两种腭裂模型中发现相似的舌异常表型、舌肌中的肌向分化标记物 MRF 家族表达下调以及 Wnt 信号通路的基因差异表达，提示了维甲酸和 Wnt5a 可能调控舌的发育。本项目首先在体内利用维甲酸诱导腭裂模型确定舌发育时空动态变化并探究舌发育异常的机制，在体外肌向诱导体系中明确了Wnt5a/CaMKⅡ对维甲酸诱导舌发育异常的调控机制；其次通过miRNAs芯片筛选与RA诱导的腭裂舌发育异常相关的差异miRNAs并进行了生物信息学分析，对筛选出的直接靶向作用于Wnt5a/CaMKⅡ的miR-27以及miR-31等的调控机制进行了深入分析。本项目在国内外首次系统研究了维甲酸诱导小鼠颌面部发育畸形中micro-RNAs 与Wnt信号分子通路的靶向调控分子机制，为深入了解环境与基因交互作用在腭裂、舌发育异常中的分子机理，为上述颌面部先天性发育畸形的防治提供科学依据。