Live cell imaging of radial glial cells of the cerebral cortex

放射状胶质细胞早期出现于神经上皮，呈高度极化结构。它们的细胞体位于脑室或脑室下区，其长放射状突起横跨神经管，止于脑髓表面，端足呈圆锥形。在发育中的大脑皮层中，它们作为神经元祖细胞和有丝分裂后神经元迁移的支架。尽管人们认为放射状胶质细胞与神经元的细胞间相互作用对引导甚至促进迁移很重要，但放射状胶质细胞在这一时期的动态行为仍然很大程度上未知。为了解决这个问题，我们开发了一个放射状胶质细胞的活细胞成像系统。我们检测了几种类型的膜锚定eYFP（增强黄色荧光蛋白），发现具有k-Ras法尼化序列的eYFP- caax均匀地标记放射状胶质细胞表面。根据Morin等人（Nat Neurosci 10, 1440, 2007）的方法，我们使用Cre-loxP系统，结合质粒载体，通过电穿孔标记有限数量的放射状胶质细胞。Cre与loxP质粒的比值为10^<-4>-10^<-5>:1，使我们能够检查每个放射状胶质细胞的形态。在胚胎第13.5天、16.5天或19天（=出生后0天）对固定制剂中标记的放射状胶质细胞进行分析表明，它们的细胞表面光滑，除了在脑膜表面附近，整个放射状突起偶尔有突起或分支。随着发育的进行，径向纤维的末端分支出现、延伸并增加。高浓度的氧气促进了标记细胞在器官型切片培养中的存活，并使我们能够在共聚焦显微镜下观察它们在16小时内的行为。这些技术上的改进将使我们能够分析神经元迁移过程中放射状胶质细胞的详细行为。

Radial glial cells arise early from the neuroepithelium and show a highly polarized structure. Their cell bodies are located in the ventricular or subventricular zone, and their long radial processes span the neural tube, ending at the pial surface with conical endfeet. In the developing cerebral cortex, they serve as neuronal progenitors and as scaffolds for migration of post-mitotic neurons. Although it is supposed that cell-to-cell interactions of radial glial cells with the neurons are important for guiding, even more promoting the migration, dynamic behavior of radial glial cells during this period remains largely unknown. To address this issue, we developed a system for live cell imaging for radial glial cells. We examined a several types of membrane anchored eYFP (enhanced yellow fluorescent protein), and found that eYFP-CAAX, which has k-Ras farnesylation sequence, uniformly labeled radial glial cell surface. According to the method for Morin et al. (Nat Neurosci 10, 1440, 2007), we applied the Cre-loxP system using a combination of plasmid vectors to label a limited number of radial glial cells by electroporation. With a 10^<-4>-10^<-5>: 1 ratio of Cre to loxP plasmids enabled us to examine each radial glial cell morphology. Analyses of labeled radial glial cells in fixed preparations at embryonic day 13.5, 16.5, or 19 (= postnatal day 0) indicated their smooth cell surface with occasional protrusions or branches throughout the radial process except near the pial surface. The terminal branches of radial fiber emerged, extended and increased in number as development proceeded. High concentration of oxygen facilitated the survival of labeled cell in organotypic slice culture and enabled us to examine their behavior over 16 hours under a confocal microscope. These technical improvements will allowed us to analyze detailed behavior of radial glial cells during neuronal migration.