Potential of neural stem cells during development.

哺乳动物的大脑皮层由六层神经元组成。脑室区域的皮质祖细胞通过连续的细胞分裂产生每一层特有的神经元。VI层的神经元是在早期阶段产生的，而较晚出生的神经元逐渐占据上层。其潜在的分子机制鲜为人知。在这项研究中，我们设计了一个系统，通过体内电穿孔和Cre介导的DNA重组在大脑皮质时空激活Notch通路。E13.5的电穿孔将DNA转移到早期的祖细胞，从而产生了低层和上层的神经元。在E13.5处强制表达一种构成活性形式的Notch(CaNotch)抑制祖细胞生成神经元，并保持祖细胞作为增殖的放射状神经胶质细胞。随后在E15.5位将Cre表达载体转染至Cre表达载体以去除caNotch后，切除caNotch的双转染细胞迁移至皮质板，并分化为上层特异性神经元。溴脱氧尿嘧啶核苷标记实验表明，Cre基因转染后神经元再生。这些结果表明，早期暂时受到Notch激活的皮质前体细胞在后期产生了神经元，但跳过了低层神经元的产生。此外，双转基因细胞即使在移植到E13.5脑中后也能产生上层神经元，这表明前体细胞的发育状态并没有因caNotch活性而停止。

The mammalian cerebral cortex is comprised of six layers of neurons. Cortical progenitors in the ventricular zone generate neurons specific to each layer through successive cell divisions. Neurons of layer VI are generated at an early stage, whereas later-born neurons occupy progressively upper layers. Its underlying molecular mechanisms are little known. In this study, we devised a system where the Notch pathway was activated spatiotemporally in the cortex by in vivo electroporation and Cre-mediated DNA recombination. Electroporation at E13.5 transferred DNA to early progenitors that gave rise to neurons of both low and upper layers. Forced expression of a constitutively active form of Notch (caNotch) at E13.5 inhibited progenitors from generating neurons and kept progenitors as proliferating radial glial cells. After subsequent transfection at E15.5 of a Cre expression vector to remove caNotch, double-transfected cells, where caNotch was excised, migrated into the cortical plate and differentiated into neurons specific to upper layers. Bromodeoxyuridine-labeling experiments showed that the neurons were born after Cre transfection. These results indicate that cortical progenitors that had been temporarily subjected to Notch activation at an early stage generated neurons at later stages, but that the generation of low-layer neurons was skipped. Moreover, the double-transfected cells gave rise to upper-layer neurons, even after their transplantation into the E13.5 brain, indicating that the developmental state of progenitors is not halted by caNotch activity.