Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya, Tokyo 156-8506, Japan.
Neuroscience. 2012 Jan 10;201:67-84. doi: 10.1016/j.neuroscience.2011.11.006. Epub 2011 Nov 11.
Pyramidal neurons of the neocortex are produced from progenitor cells located in the neocortical ventricular zone (VZ) and subventricular zone (SVZ) during embryogenesis. RP58 is a transcriptional repressor that is strongly expressed in the developing brain and plays an essential role in corticogenesis. The expression of RP58 is strictly regulated in a time-dependent and spatially restricted manner. It is maximally expressed in E15-16 embryonic cerebral cortex, localized specifically to the cortical plate and SVZ of the neocortex, hippocampus, and parts of amygdala during brain development, and found in glutamatergic but not GABAergic neurons. Identification of the promoter activity underlying specific expression patterns provides important clues to their mechanisms of action. Here, we show that the RP58 gene promoter is activated prominently in multipolar migrating cells, the first in vivo analysis of RP58 promoter activity in the brain. The 5.3 kb 5'-flanking genomic DNA of the RP58 coding region demonstrates promoter activity in neurons both in vitro and in vivo. This promoter is highly responsive to the transcription factor neurogenin2 (Ngn2), which is a direct upstream activator of RP58 expression. Using in utero electroporation, we demonstrate that RP58 gene promoter activity is first detected in a subpopulation of pin-like VZ cells, then prominently activated in migrating multipolar cells in the multipolar cell accumulation zone (MAZ) located just above the VZ. In dissociated primary cultured cortical neurons, RP58 promoter activity mimics in vivo expression patterns from a molecular standpoint that RP58 is expressed in a fraction of Sox2-positive progenitor cells, Ngn2-positive neuronal committed cells, and Tuj1-positive young neurons, but not in Dlx2-positive GABAergic neurons. Finally, we show that Cre recombinase expression under the control of the RP58 gene promoter is a feasible tool for conditional gene switching in post-mitotic multipolar migrating young neurons in the developing cerebral cortex.
新皮层的锥体神经元是在胚胎发生过程中由位于新皮层脑室区 (VZ) 和侧脑室下区 (SVZ) 的祖细胞产生的。RP58 是一种转录抑制剂，在发育中的大脑中强烈表达，在皮质发生中发挥重要作用。RP58 的表达受到严格的时间和空间限制调节。它在 E15-16 胚胎大脑皮层中表达最强，在大脑发育过程中特异性定位于皮质板和新皮层、海马体和杏仁核的 SVZ，并在谷氨酸能神经元中表达，但不在 GABA 能神经元中表达。鉴定特定表达模式背后的启动子活性为其作用机制提供了重要线索。在这里，我们展示了 RP58 基因启动子在多极迁移细胞中被明显激活，这是首次在体内分析 RP58 启动子在大脑中的活性。RP58 编码区的 5.3 kb 5'侧翼基因组 DNA 在体外和体内均显示出神经元的启动子活性。该启动子对转录因子神经基因 2 (Ngn2) 高度敏感，Ngn2 是 RP58 表达的直接上游激活剂。通过在体电穿孔，我们证明 RP58 基因启动子活性首先在 VZ 中呈针状的细胞亚群中检测到，然后在位于 VZ 上方的多极细胞积累区 (MAZ) 中迁移的多极细胞中被明显激活。在分离的原代皮质神经元中，RP58 启动子活性从分子角度模拟了体内表达模式，即 RP58 在一小部分 Sox2 阳性祖细胞、Ngn2 阳性神经元祖细胞和 Tuj1 阳性年轻神经元中表达，但不在 Dlx2 阳性 GABA 能神经元中表达。最后，我们证明，在 RP58 基因启动子控制下表达 Cre 重组酶是一种可行的工具，可用于在发育中的大脑皮质中已分化的多极迁移年轻神经元中进行条件性基因转换。
