Fig 2: YAP/TAZ are required for myelin maintenance.(A) Living eight week-old WT and Yap/Taz iDKO (Sox10-Cre-ERT2) mice 20 days post-first tamoxifen injection. Arrows indicate abnormal splayed gait. (B) Cartoon showing timeline of tamoxifen injection and time of sacrifice/ death of iDKO mice due to severity of symptoms. Purple dots: Sox10-creERT2; Yapfl/fl; Tazfl/fl; Orange dots: Plp1-creERT2; Yapfl/fl; Tazfl/fl iDKO mice. (C) Representative images of CMAPs generated in WT and iDKO mice. Right panel: Nerve conduction velocity, n = 3 mice per genotype. *p=0.0186, unpaired Student’s t-test. (D) Longitudinal cryosections of sciatic nerves of 11 week-old WT and iDKO (Sox10-Cre-ERT2) mice, 20 days after first tamoxifen injection, showing loss of YAP/TAZ (green) in iDKO but not WT SC nuclei, marked by Sox10 (red). All cell nuclei are marked by DAPI staining (blue). Asterisks mark lack of deletion of YAP/TAZ in non-SCs. n = 2 mice per genotype; two sections per mouse. (E) Transverse sciatic nerve (E1-3) and longitudinal ventral root (E4, E5) sections from 11 week old WT, iDKO (Sox10-Cre-ERT2; E2–E4) and iDKO (Plp1-Cre-ERT2; E5) mice, 20 days after first tamoxifen injection. (E1–E3) TEM of WT (E1) and iDKO (E2, E3) sciatic nerves. Axons with abnormal myelin profiles are marked with a yellow ‘a’; completely demyelinated axons are marked with a red ‘a’; myelin-laden macrophages are marked with a red ‘m’. n = 3 mice of each genotype. (E4–E5) Semi-thin ventral root sections, showing loss of myelin and loosened myelin sheaths in iDKO (Sox10-Cre-ERT2) and iDKO (Plp1-Cre-ERT2) mice. Demyelinated axons are marked by ‘a’ and myelin-filled macrophages are marked by ‘m’. Note the demyelinated internodes (marked by sets of ‘a’) contiguous with normally myelinated internodes. (F) Bar graph showing percentage of SCs immunopositive for YAP/TAZ 20 days after first tamoxifen injection, in WT and iDKO (Sox10-Cre-ERT2) sciatic nerve. (G) Quantification of demyelinating SC profiles 20 days after first tamoxifen injection, in transverse sections of WT and iDKO (Sox10-Cre-ERT2) sciatic nerve. n = 3 mice per genotype, ****p<0.0001, unpaired Student’s t-test. The following figure supplements are available for Figure 6.DOI: http://dx.doi.org/10.7554/eLife.20982.015

Fig 3: The effects of FLX on the late differentiation and senescence of oligodendrocytes in the hippocampus of APP/PS1 mice. (A) Immunofluorescence staining of SOX10+ cells in the hippocampus. Arrows (?) indicate the SOX10+ cells. Bar = 50 µm. (B) Density of SOX10+ cells in the hippocampus (x ± SD). (C) ELISA results for soluble human Aß40 and Aß42 levels (x ± SD). (D) Immunofluorescence-based morphology of Aß in the hippocampus and the number of Aß plaques with a diameter larger than 20 µm. Aß plaques were deposited in the hippocampi of APP/PS1 mice, and FLX ameliorated Aß plaque deposition. Arrows (?) indicate Aß plaques in the hippocampus. High-magnification images of Aß staining in 10-month-old APP/PS1 + NaCl and APP/PS1 + FLX mice are shown in each left corner. Bar = 500 µm. (E) Immunofluorescence staining of p16+/Olig2+ cells in the hippocampus. Arrows (?) indicate p16+/Olig2+ cells. Bar = 20 µm. (F) The ratio of p16+ oligodendrocytes to oligodendrocyte lineage cells in the hippocampus (x ± SD). *p < 0.05. **p < 0.01.

Fig 4: Inductive differentiation of hiPSCs into NCCs. a Schematic diagram of inductive differentiation. The hiPSCs were maintained in the SCM and induced the differentiation into NCCs in the NDM for 7 days. b Immunofluorescence staining of the hiPSCs before induction. c Immunofluorescence staining of the hiPSC-derived NCCs after 7 days of induction. d FACS analysis of P75 and HNK-1 dual-positive cells. e qRT-PCR analysis of the hiPSC-related genes (NANOG, OCT4, SOX2) and the NCC-related genes (P75, TFAP2A, TFAP2B, SOX9, SOX10). *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: DIRC3 induces closed chromatin at its site of expression thereby blocking SOX10 DNA binding and activating IGFBP5.ChIP assays were performed in DIRC3 depleted SK-MEL-28 and control cell lines using the indicated antibodies against either SOX10, H3K27ac or an isotype specific IgG control. (A) DIRC3 depletion was confirmed using qRT-PCR. Western blotting showed that SOX10 protein levels do not change upon DIRC3 knockdown. ACTIN was used as a loading control. (B) The indicated SOX10 binding sites were analysed by qPCR. % input was calculated as 100*2^(Ct Input-Ct IP). (C) DIRC3 depletion leads to an increase in H3K27ac levels at SOX10 bound regulatory elements within the DIRC3 locus. (D) SOX10 represses IGFBP5 expression. SOX10 was reduced in SK-MEL-28 cells using transfection of two independent siRNAs (see Fig 2C for SOX10 levels). IGFBP5 expression was quantified using RT-qPCR three days later. POLII was used as a reference gene and expression changes are shown relative to a non-targeting control (set at 1). (E) Model illustrating that DIRC3 acts locally to close chromatin and prevent SOX10 chromatin binding at melanoma regulatory elements within its locus. This leads to a block in SOX10 mediated repression of IGFBP5 and subsequent increase in IGFBP5 expression. All qPCR results are presented as mean values +/- SEM, n = 3. One-tailed student’s t-test p < 0.05 * p < 0.01 **.