Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression - PubMed (original) (raw)

Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression

Christine D Pozniak et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Overexpression of SoxE factors but not Olig genes is sufficient to drive differentiation toward the oligodendrocyte lineage. (A) Empty virus infected cells do not express O4 in EGF/FGF, whereas Sox8, Sox9, or Sox10 virus-infected cells express O4 at significant levels. In contrast, Olig1 or Olig2 expression does not lead to increased O4 staining in these culture conditions. (B) The efficiency of O4+ cell production after Sox10 expression under nondifferentiating conditions (in EGF/FGF containing medium). Results pooled from three separate experiments. Error bars are ± SEM, ***P < 0.005. (_C_) Cells infected with either Empty or SoxE-expressing retroviruses (red) cultured in 2% FBS for 48 h and stained with O4 antibody (green). _Inset_ shows Sox10-infected cells also stain with CNPase, a marker of terminally differentiating oligodendrocytes. (_D_) Cells infected with either Empty or SoxE-expressing retroviruses (red) cultured in thyroid hormones for 48 h and stained with O4 antibody (green). _Inset_ shows Sox10-infected cells also stain with CNPase, a marker of terminally differentiating oligodendrocytes. (_E_) The percentage of O4+ cells infected with Sox8, 9, or 10 cultured in serum or thyroid hormones for 48 h. Results are pooled from >3 separate experiments; error bars are ±SEM; *P < 0.05; **P < 0.02; ***P < 0.005.

Fig. 2.

Suppressor of Fused (Sufu) is a direct target of Sox10. (A) Schematic of the Sufu locus depicting the Sox10-binding site identified in the first intron of the Sufu gene (

SI Methods

). (B) Quantitative mRNA profiling using whole mouse transcriptome Affymetrix chips. Each value is shown as a ratio of the experimental value over Empty virus-infected controls. Shown are the relative expression of Sufu mRNA 48 h after infection with Sox10 virus or 24 h after infection with Mash1 or Ngn2 retroviruses that drive neurogenic differentiation in EGF/FGF containing medium. Note Mash1 and Ngn2 drive modest increases in Sufu expression, whereas Sox10 leads to a twofold decrease in expression of Sufu. Normalized intensity values of triplicate chip hybridization experiments are presented. Each sample was prepared from pools of mRNA derived from less than three separate experiments. (C) Sox10 expression leads to decreased Sufu protein levels in NPCs 24 h after plating. Note the slightly higher and broader band in the positive control lane was obtained from extracts of HEK-293T cells expressing both human and mouse HA-tagged Sufu. In contrast, NPCs only express endogenous untagged mouse Sufu. (D) Targeted ChIP for Sox10 bound to the Sufu gene at the first intronic site. Positions of the PCR primers are shown in relationship to the Sufu and Trim8 genes. The PCR yields the expected products for both Sufu and Trim8 after infection with either Empty or Sox10 virus (Input), but only Sox10-infected cells yielded a specific band for Sufu after ChIP (IP: anti-HA). Empty virus-infected cells showed no signal, and Sox10 showed no binding to the closely linked Trim8 gene. The arrows indicate the size of the expected Sufu product.

Fig. 3.

Sufu inhibits Sox10-driven OPC differentiation and endogenous oligodendrocyte differentiation. (A) shRNA-mediated knock down of mouse HA-Sufu protein (solid arrow, top bands) in GP2-293 cells. Note that the endogenous human Sufu protein is unaffected (open arrow, bottom bands). (B) Percentage of O4+ cells coinfected with Sox10 and Luciferase shRNA, Sufu, or Sufu siRNA. Transduced NPCs were cultured in EGF/FGF on laminin-coated plates for 72 h. Error bars are ± SEM, ***P < 0.005, n = 3. (C) Percentage of O4+ cells coinfected with Sox10 virus, and either Empty or Sufu virus then induced to differentiate by removing EGF/FGF and addition of serum. Error bars are ±SEM, **P < 0.01, n = 3. (D) Percentage of O4+ cells infected with Empty or Sufu virus and cultured in serum on laminin-coated plates for 72 h. Error bars are ± SEM, ***P < 0.01, n = 3.

Fig. 4.

Decreased Sufu expression significantly increases the number of oligodendrocyte precursors in vivo. (A) Western blot analysis of equal amounts of protein derived from brain lysates of wild-type (Sufu+/+) mice or mice lacking one copy of Sufu (Sufu+/−). (B) Olig2 staining in brain sections of Sufu+/+ or Sufu+/− E15.5 brains show an overall increase in Olig2-positive cells in Sufu+/− brains. Higher magnifications in Lower. (Scale bar: 100 μm.) (C) The number of PDGFRα-positive cells in the forebrain is significantly increased in mice with decreased Sufu expression (Sufu+/−) at E15.5 (Error bars are ±SD, ***P < 0.005, n = 3). (D) The increased number of PDGFRα-positive cells persists in the Sufu+/− P2 forebrain. Error bars are ± SD, **P < 0.05, n = 3.

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Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression - PubMed (original) (raw)