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Fig. 5. Pten-null granule neurons display normal migratory properties. (A) High-magnification time-lapse video microscopy revealed the migrating neurons attached to laminin-coated glass fiber. (B) Competitive migration assay showed similar migration capacity between Pten-null and control granule neurons. Representative neuron migration from reaggregate after a 24 hour incubation was shown on micrograph (top panel). Comparisons of numbers of control versus mutant neurons that migrated to each bin are shown on the histogram (bottom). A value of 1 represents an equal ratio of control and mutant neurons in a specific bin. (C) Pten-null granule neurons exhibited the typical migrating profile on organotypic cerebellar slice culture. Pten^loxp/loxp slices from P6 mice were infected with a recombinant retrovirus expressing CRE-GFP and DsRed. After 48-72 hours incubation, the infected granule neurons with nuclear GFP and cytosol DsRed expression were visualized using a confocal microscope. (D) Pten-null and wild-type granule neurons were co-cultured with cerebellar astroglia. (D, a,a') pure cerebellar glia 7 days in vitro. (b,b') Wild-type granule neurons were co-cultured with either wild-type or mutant glia for 36 hours, wild-type neurons (Tuj1) formed a close apposition with GFAP-positive mutant glial fibers during migration. (c,c') Mutant granule neurons were co-cultured with wild-type or mutant glia, and showed normal migrating profile and axon extension along with GFAP-positive astroglial fibers. Arrowheads indicate migrating neurons closely apposite with glial fibers. Scale bars: 50 µm.

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