Screening of the Myelination Factor the Lightcycler- FastStart DNA Master SYBR Green I (Roche fically in the oligoden-drocyte lineage cells. Larvae carrying the Applied Science) according to the manufacturer’s instructions. Tg(olig2:eg fp) and Tg(sox10:eg fp) transgenic reporters, which Each reaction was performed in a volume of 20 µl with final express EGFP under the control of the olig2 and sox10 promoters concentration of 1× Lightcycler-FastStart DNA Master SYBR (Shin et al., 2003; Carney et al., 2006), respectively, were examined Green 1, 3 mM MgCl2, 0.5 µM of each primer and 2 µl of first for use in the transcriptome analysis. strand cDNA mixture. Quantitative real-time PCR was carried out Expression of EGFP in OPCs and mature oligo-dendrocytes at for 40 cycles and fluorescence readings were acquired at the end several developmental stages in the Tg(olig2:egfp) and Tg(sox10: of each am-plification cycle at 72oC. Melting curve analysis was egfp) larvae was verified by fluorescence microscopy using trans- performed with continuous fluorescence acquisition from 65 to verse sections of the spinal cord. By 2 days post- fertilization (dpf ), 95oC at a temperature transition rate of 0.1oC/s to determine the sox10:EGFP+ cells are located in the gray matter of the spinal cord, amplification specificity. All reactions were performed as technical in which Hu+ neurons are located, indicating that sox10:EGFP+ duplicates. cells are undifferentiated OPCs (Fig. 1A, arrow-heads). By 5 dpf, sox10:EGFP+ OPCs have migrated to the white matter of the spi- Immunohistochemistry nal cord and differentiated into mature oligodendrocytes with Embryos were anesthetized until movement had ceased and multiple processes (Fig. 1B, arrows). Labeling of the spinal cord fixed in 4% paraformaldehyde over-night. Fixed embryos were of Tg(olig2:egfp) larvae with anti-Sox10, a marker for OPCs and mature oligo-dendrocytes, revealed that Sox10+, olig2:EGFP+ embedded in 1.5% agar with 5% sucrose blocks and equilibrated by 30% sucrose solution. Frozen blocks were sliced into 10 µm cells are located in the gray matter of ventral spinal cord at 2 dpf, sections on glass slides using cryostat micro-tome. Sections were indicating that these cells are undif-ferentiated OPCs (Fig. 1C, rinsed with PBS several times and then blocked in 2% bovine se- rum albumin with sheep serum. Embryos were processed for im- munohistochemistry, following primary antibodies were used: rabbit anti-Sox10 (1 : 250), rabbit anti-MBP (1 : 100), rabbit anti- GFP (1 : 500, Abcam) and mouse anti-HuC/D (16A11 1 : 20, Molecular Probes, Eugene, OR). For fluorescent detection of antibody labeling, we used Alexa Fluor 568-conjugated goat anti- mouse IgG, Alexa Fluor 568-conjugated goat anti-rabbit IgG, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 : 500, Molecular Probes). Photos were taken using a confocal lasers canning microscope (LSM 510 Pascal, Carl Zeiss). RESULTS AND DISCUSSION Identification of EGFP expression in the oligo-dendrocyte lineage cells of transgenic zebrafish To identify the genes involved in oligodendrocyte differentiation and myelination, we isolated OPCs and differentiated oligoden- drocytes expressing EGFP from the transgenic zebrafish and com- pared their transcriptional profiles. Therefore, the embryo stages suitable for collecting OPCs and differentiated oligodendrocytes were first identified. Previously, we have shown that olig2-expres- sing precursor cells in the pMN precursor domain of the ventral spinal cord first generate motor neurons during neuro-genesis and later produce migrating OPCs, which express olig2 continuously to the mature oligo-dendrocyte stage (Park et al., 2002a; Park and Appel, 2003; Park et al., 2004). As soon as OPCs are specified in the pMN precursor domain, sox10 starts to be expressed speci- http://dx.doi.org/10.5607/en.2011.20.2.85 Fig. 1. Identification of EGFP expression in oligodendrocyte lineage cells in transgenic zebrafish lines. All panels show transverse sections of the spinal cord, dorsal side at the top of the image. (A, B) Labeling of Tg(sox10:eg fp) larvae with anti-Hu at 2 dpf (A) and 5 dpf (B). Arrowheads indicate OPCs in the gray matter and arrows indicate differentiated oligodendrocytes in the white matter of the spinal cord. (C, D) Labeling of Tg(olig2:egfp) larvae with anti-Sox10 at 2 dpf (C) and 5 dpf (D). Arrowheads indicate olig2:EGFP+, Sox10+ OPCs in the gray matter and arrows indicate olig2:EGFP+, Sox10+ differentiated oligodendrocytes in the white matter of the spinal cord. (E, F) Labeling of TSA-treated (E) and untreated (F) Tg(olig2:egfp) larvae with anti- Sox10 at 5 dpf. Arrows indicate olig2:EGFP+, Sox10+ differentiated oligodendrocytes in the white matter of the spinal cord. www.enjournal.org 87