Indian Hedgehog b Function Is Required for the Specification of Oligodendrocyte Progenitor Cells in the Zebrafish CNS

Ihhb function is required for the specification of OPCs. A, B, Scrambled MO-injected control (A) and ihhb MO-injected (B) Tg(hsp70:ihhb-mcherry) embryos heat shocked at 20 hpf. C, Quantification of motor neurons (sMNs), OPCs, and mature oligodendrocytes (mOLs) in wild-type and ihhb MO-injected embryos (***p < 0.001). Data were obtained from 15 sections from each of 6 control and 6 ihhb MO-injected embryos. D–K, Transverse sections of spinal cord, dorsal to the top. Wild-type (D, F) and ihhb MO-injected (E, G) Tg(olig2:egfp) embryos were labeled with anti-Neurolin (D, E) and anti-Sox10 (F, G) antibodies. H, I, In situ RNA hybridization of wild-type (H) and ihhb MO-injected (I) embryos with an olig2 probe. J, K, Wild-type (J) and ihhb MO-injected (K) Tg(mbp:egfp) embryos were labeled with anti-Sox10 antibody. Arrowheads indicate olig2:EGFP⁺/Sox10⁺ OPCs in F and mbp:EGFP⁺/Sox10⁺ mature oligodendrocytes in J and K. Scale bar, 20 μm.

Ihhb function is required for neurogenesis-oligodendrogenesis switching by regulating precursor proliferation. A–F, Transverse sections of the spinal cord of Tg(olig2:egfp) embryos, dorsal to the top. Wild-type (A), ihhb MO-injected (B), heat-shocked Tg(hsp70:ihhb-mcherry);Tg(olig2:egfp) (C), shh MO-injected (D), heat-shocked Tg(hsp70:gal4;uas:shh);Tg(olig2:egfp) (E), and cyclopamine-treated (F) Tg(olig2:egfp) embryos were treated with BrdU for 20 min at 2 dpf, and labeled with anti-BrdU antibody. Dashed circles outline the spinal cord. G, H, Quantification of BrdU⁺/olig2:EGFP⁻ cells (***p < 0.001) (G) and BrdU⁺/olig2:EGFP⁺ cells (*p < 0.05). OV, Overexpression. Data were obtained from 15 sections from each of 6 control and 6 MO-injected embryos. I–N, Transverse sections of the spinal cord, dorsal to the top. (I, J) Wild-type (I) and ihhb MO-injected (J) embryos were incubated with BrdU for 20 min at 2 dpf, cultured for one more day, and then labeled with anti-BrdU and anti-Hu antibodies at 3 dpf. K, L, In situ RNA hybridization of wild-type (K) and ihhb MO-injected (L) embryos with a ngn1 probe. M, N, Labeling of wild-type (M) and ihhb MO-injected (N) Tg(olig2:dsred) embryos with anti-Isl antibody at 4 dpf. O, Quantification of BrdU⁺/Hu⁺ cells from I and J, and Isl⁺/olig2:Dsred⁺ cells from M and N. Data were obtained from 15 sections from each of 6 control and 6 ihhb MO-injected embryos. P, Real-time RT-PCR analysis to compare the expression of cell cycle regulators in wild-type, ihhb MO-injected embryos and olig2:EGFP⁺ cells FACS-isolated from wild-type and ihhb MO-injected Tg(olig2:egfp) embryos. Data are shown as the average fold change relative to the wild-type control and are normalized to β-actin expression. The results are presented as the mean ± SD from three independent experiments (***p < 0.001). Scale bar, 20 μm.