Ah-Young Chung, et al. arrowheads). Sox10+, olig2:EGFP+ cells migrated to the white matter and differentiated into mature oligodendrocytes pos- sessing multiple process by 5 dpf (Fig. 1D, arrows). These data demonstrated that in the spinal cord of Tg(olig2:eg fp) and Tg (sox10:eg fp) larvae, EGFP-expressing cells are OPCs at 2 dpf and mature oligodendrocytes at 5 dpf. Thus, OPCs and mature oligodendrocytes can be obtained by sorting EGFP+ cells from Tg (olig2:egfp) and Tg(sox10:egfp) 2 dpf and 5 dpf larvae, respectively. To identify the genes specific to oligodendrocytes, a comparison of transcriptional profiles of normal and oligodendrocyte- deficient larvae could be con-ducted. Previously, oligodendrocyte development was shown to require histone deacetylase (HDAC) activity (Marin-Husstege et al., 2002; Cunliffe and Casaccia- Bonnefil, 2006; Ye et al., 2009), and treatment with the HDAC inhibitor Trichostatin A can block formation of oligodendrocytes in zebrafish (Tsai et al., 2006). Consistent with previous reports, we found that treatment of Tg(olig2:egfp) embryos with TSA from 36 hours post fertilization (hpf ) to 5 dpf blocked migration of Sox10+, olig2:EGFP+ oligodendrocytes, which are located in the white matter of the spinal cord in the normal 3 dpf larvae (Fig. 1E and F, arrows indicate mature oligoden-drocytes). These data indicated that comparison of transcriptional profiles of EGFP+ cells from TSA- treated and untreated Tg(olig2:egfp) larvae can be used to identify genes expressed specifically in oligodendrocytes. Microarray-based transcriptome analysis to identify genes involved in oligodendrocyte differentiation On the basis of the above data, OPCs and mature oligodendro- cytes were purified from the dis-sociated cells of Tg(olig2:egfp) and Tg(sox10:egfp) larvae at 2 dpf and 5 dpf, respectively, using FACS. EGFP+ cells were also purified from TSA-treated and untreated 3 dpf Tg(olig2:egfp) larvae. Prior to sorting of EGFP+ cells, dissociated cells from the Tg(olig2:egfp) and Tg(sox10:egfp) larvae contained 20% EGFP+ cells (Fig. 2A~C). After sorting, an average of 98% of the sorted cells was EGFP+ OPCs or mature oligodendrocytes (Fig. 2D~F). To confirm the purity of the sorted cells, total RNA was isolated from EGFP+ mature oligo- dendrocytes and semi-quantitative RT-PCR was performed with several PCR primers specific to mature oligodendrocytes, neurons, and blood vessels. Mature oligodendrocyte specific genes were successfully amplified by RT-PCR, but neuron and blood vessel specific genes were not. These data indicate that oligodendrocyte lineage cells were specifically isolated by FACS (Fig. 2G). To compare the transcriptional profiles of OPCs and mature oligodendrocytes, we purified RNA from the sorted cells and synthesized dye-labeled cDNAs, which were hybridized to microarray gene chips. A total of 19,956 transcripts were examined with the Agilent zebrafish gene expression microarray. Genes up- regulated by 2.5-fold in mature oligodendrocytes were identified as genes required for oligoden-drocyte maturation and genes down- Fig. 2. Sorting of EGFP+ oligodendrocytes from transgenic zebrafish using FACS. Differential interference contrast images (DIC; A, D), fluorescence images (B, E), and combined DIC and fluorescence images of the same samples (C, F). (A~C) Tg(sox10:egfp) larvae were trypsinized to disso- ciate them into single cells at 5 dpf. (D~F) EGFP+ oligodendrocytes were sorted from Tg(sox10:egfp) larvae by FACS. More than 98% of the sorted cells were EGFP+ oligodendrocytes. (G) Semi-quantitative RT-PCR with RNA purified from the sorted 5 dpf sox10:EGFP+ cells and PCR primers specific to genes expressed in mature oligodendrocytes (sox10, mbp, gfp), neurons (Hu), and blood vessels (flk). β-actin was used as a loading control. 88 www.enjournal.org Fig. 3. Comparison of the transcriptional profiles obtained from three different algorithms. (A, B) Venn diagrams show the numbers of genes detected by three different algorithms, including the numbers of overlapping genes. (A, B) The intersection of the three databases contains 32 genes that were up- regulated in differentiated oligodendrocytes (A) and 19 genes that were down-regulated in differentiated oligodendrocytes (B). (C) Microarray profiles were validated by examining the expression profiles of genes known to be specifically expressed in differentiated oligodendrocytes, including mpz, plp1b, mag, cldnk and flj13639. http://dx.doi.org/10.5607/en.2011.20.2.85