Smek蛋白调控Wnt/Ryk信号通路影响胎鼠大脑皮层早期发育的分子机制

Abnormal of Wnt signaling during the early development of embryonic cerebral cortex has been implicated in neurodevelopmental disorders. The Ryk protein was identified as a noncanonical Wnt receptor, and its activity is required for corticogenesis. However, the mechanisms whereby Wnt/Ryk signaling directs neural stem cell self-renewal and differentiation are still poorly understood. Our preliminary data show that the nuclear protein Smek interact with Ryk ICD, and Smek overexpression increases Ryk ICD nuclear localization and neurogenesis. Therefore we hypothesize that Smek acts as a key regulator of Wnt/Ryk signaling pathway during neurogenesis. In this project, we will utilize RNAi technology, in utero electroporation, and gene knockout mouse model to determine 1) if Smek synergizes with Ryk ICD to promote neurogenesis; 2) if Smek regulates Ryk ICD nuclear localization and their functions in Wnt/Ryk/Smek pathway; 3) how the Ryk ICD/Smek complex regulates transcription of target genes downstream of Ryk. The results gained from the proposed studies will elucidate Smek function in the development of embryonic cerebral cortex and the molecular mechanisms of Wnt/Ryk/Smek signaling pathway during neurogenesis, which will lay a foundation for revealing the pathogenic mechanisms of associated neurodevelopmental disorders, and provide the theoretical basis for preventing and treating neurodevelopmental disorders.

胚胎大脑皮层早期发育中Wnt信号通路异常与神经发育障碍性疾病(NDDs)密切相关，Ryk作为Wnt非典型受体，其活性对皮层形成十分重要。然而人们对Wnt/Ryk信号通路调控脑皮层早期发育中神经干细胞自我更新和分化的分子机制知之甚少。本课题前期研究结果表明：Smek为Ryk相互作用蛋白，其过表达增加Ryk-ICD核定位和促进神经形成，进而提出假设：Smek蛋白为神经形成中Wnt/Ryk信号通路的关键调控因子。本项目将采用RNAi技术，子宫内电穿孔技术，和基因敲除小鼠模型，从离体和在体两方面，研究Smek与Ryk在神经形成中的协同作用；研究Smek调控Ryk ICD核定位机理；以及Ryk ICD/Smek复合物调控Ryk靶基因的转录。本研究将明确Smek在胚胎脑皮层发育过程中的作用机制，阐明Wnt/Ryk/Smek信号通路，为揭示NDDs发病机理奠定基础，为NDDs防治提供理论依据。

Smek基因作为神经系统发育的重要调控因子，在神经干细胞分化过程起着重要作用。本项目首先建立了小鼠神经干细胞贴壁培养与悬浮培养两种细胞培养方法及平台，为后续神经干细胞的研究打下技术基础；建立了神经干细胞含ploy(A)尾RNA以及去除rRNA的总RNA文库构建方法，并进行了野生型和Smek敲除型神经干细胞分化过程中三个时间点转录组的测序及其分析。研究了小鼠神经干细胞分化过程中野生型和Smek敲除型基因可变剪接事件的变化情况及建立了生物信息分析平台，为从系统生物学角度研究小鼠神经发育打下基础；研究了小鼠神经干细胞分化过程中野生型和Smek敲除型lncRNA基因对神经干细胞分化的影响情况及建立了生物信息分析平台，为从系统生物学角度研究小鼠发育过程中非编码RNA的功能打下基础。