脊髓发育过程中Pax3/Pax7参与调控神经细胞分化和轴突生长的分子机制研究

Transcription factors Pax3 and Pax7 (Pax3/7) play importmant roles during vertebrate development. Our previous study showed that abnormal expression of Pax3/7 results in disorders of neural cell aggregation and axon pathway finding during chicken embryonic spinal cord development. More analysis explored that Pax3/7 are involved in Wnt/beta-catenin and Sonic Hedgehog（Shh）signaling pathways, aslo related to the expression and distribution of cadherins, however, little is known about their regulation mechanism. According to above, we hypothesize that Pax3/7 may regulate cadherin expression through Wnt/beta-catenin and Shh signaling pathways to affect neuron differentiation and axon pathway finding during the spinal cord development. In this study, we aim to investigate the misexpression of Pax3 and Pax7 in the developing embryonic spinal cord with in vivo electroporation combining with CRISPR-Cas9 system, to analyze neuron differentiation, migration and aggregation with spinal cord slice, neuron culture and real-time observation, and to confirm the involved molecules and signaling pathways with transcriptome sequencing analysis. Finally, we will demonstrate the function and regulation of Pax3/7 at four levels, living embryo - spinal cord tissue slice - cells - molecules with different methods, during the spinal cord development. Transplatation of Pax3/7 positive neural cells will be used to treat mouse spinal cord injury. This project will provide experimental data to reveal functional mechanism of Pax3/7 and their potential clinical application.

转录因子Pax3和Pax7（Pax3/7）在脊椎动物发育中功能重要。我们前期研究显示在鸡胚脊髓中异常表达Pax3/7导致神经细胞聚集和轴突路径障碍，且与Wnt/β-catenin和Sonic Hedgehog（Shh）通路及钙粘蛋白表达分布相关联，但机制不明。据此提出Pax3/7相互作用通过Wnt/β-catenin和Shh信号通路调控钙粘蛋白表达，进而影响发育脊髓中神经元分化和轴突路径选择。本研究将通过电转基因技术结合CRISPR-Cas9体系改变Pax3/7表达，采用脊髓片和神经元培养及实时观察等技术分析神经元分化迁移和轴突生长、OpenBook分析细胞聚集和轴突投射等现象，通过组学分析证实参与的分子与信号通路，从活体-组织-细胞-分子层次研究Pax3/7在脊髓发育中的功能和调控，尝试Pax3/7阳性神经细胞移植修复脊髓损伤，将为阐明Pax3/7的作用机理及其潜在临床应用提供实验资料。

转录因子Pax3和Pax7(Pax3/7)在脊椎动物发育中功能重要。本课题首先通过活体电转技术在鸡胚脊髓中异常表达Pax3，初步证实Pax3参与调控神经元轴突生长及连合神经元纤维投射。过表达Pax3抑制Pax7的表达，并且Pax7抑制表达后能够抑制神经元轴突的形成，Pax3和Pax7共转结果显示Pax7超表达可以挽救Pax3超表达缺陷，暗示Pax3通过Pax7来调控神经元轴突长度和神经元迁移。Shh在脊髓中过表达影响神经纤维投射，同时抑制Pax3和Pax7的表达，表明Shh是通过Pax3和Pax7调控神经元轴突生长。此外，我们还发现，Pax3和Pax7在脊髓发育过程中与Wnt/-catenin，以及钙粘蛋白家族有密切联系。本课题为进一步研究Pax3/Pax7在神经系统中的功能、揭示脊髓发育及脊髓神经纤维网路形成的分子机制等方面奠定实验和理论基础，也为临床治疗脊髓损伤提供新的策略。