fz8a expression revealed by in situ RNA hybridization. A,B: Dorsal views of the trunk region of whole embryos, anterior to the left. Arrowheads and brackets indicate expressions in the prechordal plate and medial neuroectoderm, respectively. C: Transverse sections, dorsal to the top, through the trunk region. Ventral neural keel (nk) cells, overlying notochord (nc), started to express fz8a at 14 hpf. D: Lateral view of the spinal cord of whole embryos, dorsal to the top and anterior to the left. Brackets indicate the expression of fz8a in the ventral spinal cord cells. E-H: Transverse sections of the spinal cord, dorsal to the top. E, F: fz8a-expressing cells occupy the ventral neural precursor cells. G: fz8a-expressing cells incorporate BrdU, as indicated by anti-BrdU antibody staining (red color), indicating that they are proliferating precursors. Arrows indicate fz8a⁺, BrdU⁺ neural precursor cells. H: fz8a-expressing cells confer olig2⁺ precursors, which are marked by EGFP fluorescence in the Tg(olig2:egfp) embryo. Arrows indicate fz8a⁺, olig2⁺ precursor cells in the ventral spinal cord.

fz8a function is not required for dorso-ventral patterning of the spinal cord. A, B: Tg(hsp70:fz8aCRDTM-egfp) embryos were heat shocked at 18 hpf. A: Control embryos had high levels of EGFP-fluorescence in the whole embryos. B: Embryos injected with fz8a MO had low levels of EGFP fluorescence, suggesting that fz8a MO works specifically to knock-down fz8a expression. C-F: Transverse sections of the spinal cord of Tg(olig2:egfp) embryos. C, E: Control embryos that show olig2-EGFP and iro3 expression (C), or olig2-EGFP and nk2.2 expression (E), revealed by in situ RNA hybridization. D, F: Tg(olig2:egfp) embryos injected with fz8a MO and hybridized by iro2 (D) and nk2.2 (F) RNA probes.

fz8a function is required for motor neuron and oligodendrocyte development. A, B: Lateral views of spinal cords hybridized with an isl-2 RNA probe at 18 hpf, dorsal to the top and anterior to the left. A: Control embryos. B: fz8a MO-injected embryos. Arrowheads indicate primary motor neurons in the ventral spinal cord. C-F: Transverse section of the spinal cord of Tg(olig2:egfp) embryos, dorsal up. C, D: Combined anti-Neurolin (red fluorescence) and olig2:EGFP labeling in control embryos (C), and fz8a MO-injected embryos (D). E, F: Labeling with an anti-Sox10 antibody to mark OPCs (red fluorescence) in control (E) and fz8a MO-injected (F) Tg(olig2:egfp) embryos. G, H: Side views of whole-mount 3-dpf embryos, dorsal up and anterior to the left. G: Control Tg(olig2:egfp) embryo showing numerous olig2:EGFP⁺ OPCs in the dorsal spinal cord (brackets). H: fz8a MO-injected Tg(olig2:egfp) embryo. Only a few OPCs are evident in the dorsal spinal cord (brackets). I, J: Quantification of Neurolin⁺ secondary motor neurons (SMNs) (I) and Sox10⁺ OPCs (J) at 48 hpf in control (cont) and fz8a MO-injected (FzMO) embryos. Bars indicate the average number of cells per transverse section. Data were obtained from 10 sections from each of 5 control and 5 fz8a MO-injected embryos.

fz8a function is required for the proliferation and spatial organization of radial glia in the ventral spinal cord. A-F: Transverse sections of spinal cords of Tg(olig2:egfp) embryos, dorsal up. A, B: Labeling of control (A) and fz8a MO-injected (B) embryos with an anti-BrdU antibody (red fluorescence) to detect proliferating cells. Arrows indicate olig2-expressing BrdU⁺ precursors. C-F: Combined anti-Zrf-1 (red fluorescence) and olig2:EGFP labeling of wt control (C, E, same section) and fz8a MO-injected (D, F, same section) embryos. fz8a MO-injected embryo shows disorganized radial glia in the ventral spinal cord (brackets). Arrows indicate processes of olig2⁺ radial glial cells labeled by olig2:EGFP and anti-Zrf-1 antibody staining (E). G: Quantification of BrdU⁺ proliferating cells at 24 hpf in control (cont) and fz8a MO-injected (FzMO) embryos. P < 0.0001 by Student's t-test for Fz8a domain. H: Quantification of the number of Zrf-1⁺ radial glial processes at 3 dpf in control (cont) and fz8a MO-injected (FzMO) embryos. Bars indicate the average number of cells per transverse section. Data were obtained from 10 sections from each of 8 control and 8 fz8a MO-injected embryos.

Down-regulation of Wnt signaling is required for the maturation of OPCs. A-I: Transverse sections of spinal cord, dorsal up. A-C: Tg(olig2:egfp) embryos labeled with an anti-Sox10 antibody to detect undifferentiated OPCs at 2 dpf. Arrows indicate Sox10⁺, olig2:EGFP⁺ OPCs in control (A), Bio-treated (B), or LiCl-treated (C) embryos from 24-48 hpf. D-F: Combined anti-Hu and anti-Sox10 labeling of control (D), Bio-treated (E), or LiCl-treated (F) embryos from 2-4 dpf. Arrowheads indicate differentiated oligodendrocytes in the white matter (D) and arrows indicate undifferentiated OPCs near the ventricle (E, F). G-I: TUNEL staining of control (G), Bio-treated (H), or LiCl-treated (I) embryos from 2-4 dpf. J-L: Side views of Tg(olig2:egfp) embryos, dorsal to the top and anterior to the left. J: Control embryo has numerous olig2:EGFP⁺ oligodendrocytes in the dorsal spinal cord (brackets). Bio-treated (K) and LiCl-treated (L) embryos from 2 dpf show only a few olig2:EGFP⁺ cells in the dorsal spinal cord (brackets) at 4 dpf. M-O: Lateral views of embryos hybridized with dm-20 RNA probes, which label mature oligodendrocytes at 4 dpf. M: Control embryos have many dm-20⁺ mature oligodendrocytes in the spinal cord (bracket). Bio-treated (N) and LiCl-treated (O) embryos have few cells marked by dm-20, suggesting that OPCs fail to differentiate into mature oligodendrocytes (brackets).

Specificity test of Fz8a MO by injection of 4misfz8a MO. All panels are transverse sections of the spinal cords of Tg(olig2:egfp) embryos, dorsal up. A, C: Wild-type embryos labeled with anti-Sox10 (A) and anti-Zrf-1 (C) antibodies to detect OPCs and radial glial processes. B, D: Embryos injected with the four base pair mismatched control morpholino for fz8a (4misfz8a MO) and labeled with anti-Sox10 (B) and anti-Zrf-1 (D) antibodies.

Bio and LiCl activate canonical Wnt signaling. Treatment of Bio- (B) or LiCl (C) at 1-cell-stage embryos shows nuclear translocation of membrane-localized β-catenin at 4 dpf, indicating that Wnt signaling is activated. Control embryo (A) shows membrane-localized β-catenin.

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