时空调控Wnt/β-catenin信号通路在小鼠前庭感受器平面细胞极性形成中的机制研究

The vestibular organs are a unique model system for studying planar cell polarity (PCP) because polarization develops at three different anatomical scales in the same sensory organ. The polarity abnormal of the vestibular organs maybe involved in the incidence of vertigo. Our previous study showed that activation/inhibition of canonical Wnt signaling pathway resulted in the misorientation of the utricle stereociliary bundle in vitro and in vivo. Thus we speculate that the canonical Wnt signaling pathway may regulate the formation of PCP in vestibular organs. However, In which development stage and tissue how to regulate PCP formation mechanism is still unknown. In this study, we use the utricle as a model system and explored the mechanisms of canonical Wnt signaling pathway in PCP regulation..①In vitro: Activation/inhibition of canonical Wnt signaling pathway in different embryonic stages, We screen the Wnt related genes related with PCP formation in different utricle maculae parts using RNA-seq methods..②In vivo, We explore the mechanisms of the Wnt pathway in the PCP regulation based on temporal (different embryonic stages)and spatial(precursor cells、hair cells and supporting cells) control of Wnt/β-catenin signaling pathway using the different Cre recombinase transgenic mice models. .③Mechanism exploration: One, in vivo We further verify whether the canonical Wnt pathway genetic interactions with PCP protein(Vangl2) exerts the roles in vestibular PCP formation; the other, in vitro we further validate whether the non-canonical Wnt/PCP signaling pathway has synergistic effect withβ-catenin involved in PCP formation ..The goal of this study is to establish the experimental foundation for regulating the polarity of new regenerated hair cells, which results in the auditory and vestibular function recovery.

前庭感受器是研究平面细胞极性（PCP）最佳器官模型，极性排列紊乱与眩晕发生密切相关。前期研究发现激活/抑制Wnt/β-catenin信号，可导致前庭器官极性紊乱。由此推测经典Wnt通路调控前庭器官PCP的发生。而不同发育时期和部位Wnt信号的调控功能与内在机制仍不明确。本研究旨在揭示经典Wnt信号调控前庭PCP形成机制。拟通过：①体外研究：在胚胎发育不同阶段干预Wnt信号，RNA-seq检测筛选不同部位影响极性改变的基因。②体内实验：通过Cre重组酶转基因小鼠在发育不同时期和部位（前体细胞、毛细胞和支持细胞）时空调控β-catenin激活/抑制Wnt信号，观察极性表型改变。③机制探讨：通过突变鼠杂交实验，验证Wnt信号是否与PCP核心蛋白发生作用；通过体外干预验证是否非经典Wnt/PCP信号参与PCP的调控。从而初步阐明Wnt调控PCP的内在机制，为新生毛细胞的极性重排研究奠定理论基础。

前庭感受器是研究平面细胞极性（PCP）最佳器官模型，极性排列紊乱与眩晕发生密切相关。前期研究发现激活/抑制Wnt/β-catenin信号，可导致前庭器官极性紊乱。由此推测经典Wnt通路调控前庭器官PCP的发生。而不同发育时期和部位Wnt信号的调控功能与内在机制仍不明确。本研究旨在揭示经典Wnt信号调控前庭PCP形成机制。我们主要通过Cre重组酶转基因小鼠在发育不同时期和部位（前体细胞、毛细胞和支持细胞）时空调控β-catenin激活/抑制Wnt信号，观察极性表型改变。在前庭感觉上皮（以椭圆囊和球囊为例）毛细胞前体细胞中抑制经典wnt/β-catenin信号途径，可以观察到椭圆囊和球囊大小和单位面积上的毛细胞数量减少，极性分界线仍然存在，但是在极性分界线两侧可见部分毛细胞极性排列紊乱。在小鼠毛细胞中条件性过表达β-catenin可以发现毛细胞排列发生紊乱，并且极性蛋白Prickle的表达位置从正常的毛细胞内侧转变为在支持细胞之间出现异常表达。在Foxg1+细胞中条件性敲除β-catenin可以导致小鼠P1时死亡，取胚胎期18.5天的胚胎发现小鼠头面部和内耳发育不良。在支持细胞中抑制经典wnt/β-catenin信号途径，未发现小鼠前庭感觉上皮毛细胞排列和极性分界线异常。结论：在发育早期毛细胞前体细胞和毛细胞中敲除β-catenin可以引起内耳耳蜗和前庭的发育异常，感觉上皮变小，毛细胞变少，纤毛发育幼稚，部分耳蜗和前庭毛细胞单个细胞极性发育异常。我们在此项目中详细研究了不同发育时期和部位激活/抑制经典wnt/β-catenin信号途径来观察前庭器官的发育和平面细胞极性的分布情况，更好地理解经典wnt/β-catenin信号在前庭器官发育过程中的作用，为今后前庭毛细胞再生恢复平衡功能提供理论基础。