| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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.
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