FACS microarray analysis of radially migrating cortical neurons

During the developmental formation of 6-layered neocortical structure of mammalian cerebral cortex, newborn excitatory neurons depart the ventricular zone and migrate toward the pial surface. At a middle stage of cortical development, newly differentiated postmitotic neurons adopt a multipolar shape (MP), and exhibit a random non-directional migration in the intermediate zone (multipolar migration). When these neurons pass through the subplate layer (SP), they convert to a bipolar shape (BP), and then migrate radially toward the pial surface (locomotion). In order to elucidate the molecular mechanisms of such neuronal migration, we performed a gene expression profiling of newborn excitatory neurons during their migration processes. After in utero electroporation of GFP expressing plasmid into E14 mouse cortex, GFP-positive cells were collected using FACS sorting method after one, two, or three days of electroporation. RNAs of collected GFP-positive cells at each day were purified and applied to microarray analyses. gene-ontology and pathway analyses revealed that genes encoding synaptic proteins, receptors and ECM-related proteins were up-regulated as the migration proceeds. On the other hand, genes encoding cell cycle regulation and immune-related proteins were down-regulated. We will discuss the relationship between the migration mode and the transition of the gene expression. pCAGGS-EGFP was electroporated in utero into the mouse embryonic cortices at E14.5, and the brains were collected at E15.5, E16.5, and E17.5. GFP-positive dorsal cortical areas were dissected out under a dissection microscope with a fluorescent lamp. GFP-positive cortical cells were dissiciated with papain and subjected to FACS cell sorter BD FACS Aria III . Cells prepared from the brains not electroporated with pCAGGS-EGFP were used as the negative controls. We performed FACS sorting 5-9 times independently (9 times for E15.5, 5 times for E16.5, and 7 times for E17.5) to pool approximately 2×105 GFP-positive cells in total for each time point.