gfap⁺ cells are radial glia in embryonic and postembryonic zebrafish spinal cord. All images are transverse sections of spinal cords, dorsal to top. A,B: Spinal cords of 36 hours postfertilization (hpf) Tg(gfap:GFP) embryos labeled by gfap (A) and egfp (B) RNA in situ hybridization. C: gfap RNA expression at 36 hpf. D: Glial fibrillary acidic protein (GFAP) expression detected by immunocytochemistry at 48 hpf. Brackets in A-D mark ventral spinal cord cells that do not express the endogenous gfap gene or the reporter transgene. Arrowheads mark floor plate. E-G: Anti-Zrf-1 antibody labeling (red) and enhanced green fluorescent protein (EGFP) fluorescence (green) of Tg(gfap:GFP) animals at 3 days postfertilization (dpf), 7 dpf, and 3 months. Numerous Zrf-1⁺ EGFP⁺ radial glia appear as yellow fibers. Arrows indicate Zrf-1⁺ GFAP^- radial glial cells in ventral most spinal cord. H: Anti-brain-lipid-binding protein (BLBP) antibody (red) labeling of 3-month-old adult Tg(gfap:GFP) fish. BLBP⁺ EGFP⁺ radial glia appear yellow. Arrows indicate BLBP⁺ EGFP^- radial fibers in ventral spinal cord. Scale bar = 20 μM in A-F, 80 μM in G,H.

gfap⁺ radial glial cells divide and produce neurons. All images are transverse sections of spinal cords obtained from Tg(gfap:GFP) embryos, dorsal up. Enhanced green fluorescent protein (EGFP) fluorescence is shown as green. A,B: Bromodeoxyuridine (BrdU) labeling (red) at 24 hours postfertilization (hpf; A) and 36 hpf (B). C,D: Anti-Hu antibody labeling (red) at 24 hpf (C) and 48 hpf (D). Arrows indicate EGFP⁺ cells with relatively weak Hu labeling, which probably are newly born neurons. Scale bar = 20 μM.

gfap⁺ cells produce motor neurons and OPCs. All panels show transverse sections of spinal cord with dorsal upward. A-D: Tg(olig2:DsRed2);Tg(gfap:GFP) embryos at 1 dpf (A,B) and 2 dpf (C,D). Arrows and arrowheads indicate DsRed2⁺ EGFP⁺ cells near the central canal and pial surface, respectively. E: Arrows mark Isl⁺ EGFP⁺ motor neurons at 1 dpf. F: Neurolin⁺ EGFP⁺ secondary motor neurons (yellow) at 2 dpf. G,H: Arrowheads indicate Sox10⁺ EGFP⁺ OPCs at 2 dpf. Scale bar = 20 μM.

Expression of notch and delta genes in spinal cord of late embryonic and early postembryonic zebrafish. All panels show in situ RNA hybridized, transverse sections of spinal cord, dorsal up. A-D: A 2 days postfertilization (dpf) late embryonic stage. E-H: A 3 dpf early postembryonic stage. Scale bar = 20 μM.

Conditional inhibition of Notch signaling before OPC specification produces excess secondary motor neurons at the expense of OPCs. All images show transverse sections of spinal cord, dorsal to top. A-D: Control embryos. E-H: Tg(hsp70l:dnSu(H)myc) embryos heat-shocked at 30 hours postfertilization (hpf). Inhibition of Notch signaling before OPC specification blocks formation of Sox10⁺ OPCs (A,E,I) and produces excess secondary motor neurons (B,F,J). Arrows mark OPCs and secondary motor neurons and the small, dashed circle marks the area around the central canal, which is not occupied by motor neurons, in control embryos. The medial portion of the spinal cord, which does not have Hu⁺ neurons in control embryos (dashed circle, C) is filled with neurons in Notch-inhibited embryos (G). Pulse labeling with bromodeoxyuridine (BrdU, red) reveals that more dividing spinal cord cells adopt Isl⁺ motor neuron fates in Notch-inhibited embryos relative to controls (D,H,K). The quantitative data were obtained from sections of six control and six heat-shocked embryos for each experiment. Error bars represent SEM. Scale bar = 20 μM.

Notch signaling is required to maintain radial glial cells during embryonic development. All panels show transverse sections of spinal cord with dorsal upward. A-C: Tg(olig2:egfp) larvae labeled with anti-Zrf-1 antibody (red) at 6 days postfertilization (dpf). Control (A), DAPT treatment begun at 32 hours postfertilization (hpf). (B), and DAPT treatment begun at 50 hpf (C). Arrows indicate Zrf-1⁺ enhanced green fluorescent protein-positive (EGFP⁺) radial glial cells, and arrowheads indicate EGFP⁺ oligodendrocyte lineage cells. D-F: A 3 dpf Tg(hsp70l:GAL4);Tg(UAS:myc-Notch1a-intra) larvae labeled with Zrf-1 antibody; control (D), heat induction at 24 hpf (E), and heat induction at 50 hpf (F). G,H: A 3 dpf larvae probed to detect her4 expression by in situ RNA hybridization. Control (G) and DAPT treatment begun at approximately 50 hpf (H). Scale bar = 20 μM.