Epha4基因通过FGF信号通路调节大脑皮层发育

Epha4 gene regulates corticogenesis through FGF signaling

目的阐明Epha4基因在大脑皮层发育早期放射性胶质细胞(RGCs)命运决定中的功能。方法利用Cre-loxp技术分别在孕龄为11.5 d或13.5 d的小鼠皮层细胞中敲掉Epha4基因。根据小鼠的基因型分为3组:对照组、Nestin;Epha4fx/fx及GFAP;Epha4fx/fx,分别通过尼氏染色、免疫荧光共染色及免疫印迹分析了突变小鼠大脑皮层表现型、大脑皮层RGCs增殖和分化及皮层细胞信号通路。组间比较利用F检验进行统计学分析。结果尼氏染色结果表明,与正常新生小鼠皮层(709±30)μm相比,在孕龄11.5 d Epha4基因敲除新生小鼠大脑皮层厚度减少至(475±66)μm,差异具有统计学意义(t=7.252,P<0.05),而孕龄13.5 d Epha4基因敲除新生小鼠大脑皮层厚度(727±37)μm,差异无统计学意义。Brd U标记及免疫荧光共染色结果表明,在孕龄14.5 d两种突变小鼠中RGCs增殖能力减弱(46±1)%vs Epha4fx/fx(58±2)%,t=10.72,P<0.05;GFAP;Epha4fx/fx(50±2)%vs Epha4fx/fx(58±2)%,t=5.575,P<0.05),而向神经元分化能力增强(Nestin;Epha4fx/fx(34±5)%vs Epha4fx/fx(25±1)%,t=4.269,P<0.05;GFAP;Epha4fx/fx(35±2)%vs Epha4fx/fx(25±1)%,t=12.48,P<0.05。Western blotting分析表明分离的皮层细胞经成纤维生长因子(FGFs)作用时ERK及FRS2α磷酸化减弱。结论 Epha4基因决定大脑皮层发育早期RGCs的命运,这一过程是通过FGFs信号通路完成的。这些发现为临床治疗脊髓损伤及阿尔茨海默症等神经疾病提供了理论依据。

Objective To illuminate the function of Epha4 gene in RGC cell fate determination during early corticogenesis. Methods Epha4 gene was deleted in cortical cells at E11.5 or E13.5 using Cre-loxp method. The mice were divided into 3 groups： Group A （control mice）, Group B （Nestin., Epha4fx/fx mutant mice） and Group C （GFAP; Epha4fx/fx mutant mice）. Nissle staining was used to analyze the morphological changes of the mutant mice. Co-immunofluoresence was used to determine the proliferation and differentiation of RGC. Western blotting was used to detect the signaling pathway in the mutant mice. And the statistical analysis was achieved using Student＇s t-test. Results Nissle stainingshowed the size of the cerebral cortex at P0 was smaller than that of controls when Epha4 was deleted at Ell.5 （475±66 μm vs 709±30 μm, P 〈 0.05） but not when it was deleted at E13.5 （727±37 μm vs 7094-30μm, P 〉 0.05）. BrdU labeling followed by co-immunofluoresence showed the proliferation ability of RGC decreased （Nestin; Epha4fx/fx 46±1% vs Epha4fx/fx 58±2%, P 〈 0.05; GFAP; Epha4fx/fx 50±2% vs Epha4fx/fx 58±2%, P 〈 0.05）, while the differentiation ability of RGC increased （Nestin; Epha4fx/fx 34±5 % vs Epha4fx/fx 25±1%, P 〈 0.05; GFAP; Epha4fx/fx 35±2% vs Epha4fx/fx 25±1%, P 〈 0.05）. Westem blotting showed cortical cells from both deletion mutants revealed lower phosphorylation of ERK and FRS2 a in the presence of FGF. Conclusion Epha4 gene, in cooperation with an FGF signal, contributes to function in RGC cell fate determination during early corticogenesis. These findings provide important molecular evidence for the clinical treatment of neurodegenerative diseases such as spinal cord injury and Alzheimer disease.