Developmental Biology 302 (2007) 683–693 www.elsevier.com/locate/ydbio

Genomes & Developmental Control Induction of differentiation by Olig2 and Sox10: Evidence for reciprocal interactions and dosage-dependent mechanisms

Zijing Liu a, Xuemei Hu a, Jun Cai a, Ben Liu a,b, Xiaozhong Peng b, ⁎ Michael Wegner c, Mengsheng Qiu a,

a Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA b The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China c Institut fuer Biochemie, Universitaet Erlangen-Nuernberg, Erlangen, Germany Received for publication 28 June 2006; revised 2 October 2006; accepted 4 October 2006 Available online 10 October 2006

Abstract

Recent studies have suggested that oligodendrocyte development is likely to be under the control of a hierarchy of lineage-specific transcription factors. In the developing mouse spinal cord, expression of Olig2, Sox10 and Nkx2.2 is sequentially up-regulated in cells of oligodendrocyte lineage. These transcription factors play essential roles in oligodendrocyte specification and differentiation. However, the regulatory relationship and functional interactions among these transcription factors have not been determined. In this study, we systematically investigated the function and hierarchical relationship of Olig2, Sox10 and Nkx2.2 transcription factors in the control of oligodendrocyte differentiation. It was found that over-expression of Olig2 is sufficient to induce Sox10, Nkx2.2 and precocious oligodendrocyte differentiation in embryonic chicken spinal cord. Sox10 expression alone is also sufficient to stimulate ectopic oligodendrocyte differentiation and weakly induce Nkx2.2 expression. Although genetic evidence indicated that Sox10 functions downstream of Olig2, Sox10 activity can modulate Olig2 expression. In addition, we presented evidence that the control of oligodendrocyte differentiation by Olig2, Sox10 and Nkx2.2 is a dosage- dependent developmental process and can be affected by both haploinsufficiency and over-dosage. © 2006 Elsevier Inc. All rights reserved.

Keywords: Spinal cord; ; In ovo electroporation; Mutation; Myelin genes; Nkx2.2

Introduction sequentially generated (Richardson et al., 2000; Spassky et al., 2000; Miller, 2002). At slightly later stages, a small number of Recent studies have identified a large number of transcription OPCs are also generated from the dorsal spinal cord independent of factors that are selectively expressed in cells of oligodendrocyte Shh signaling (Cai et al., 2005; Fogarty et al., 2005; Vallstedt et al., (OL) lineage at specific stages of cellular development (Rowitch, 2005). It is estimated that approximately 10–15% of mature OLs in 2004). Mounting evidence suggests that oligodendrocyte devel- the spinal cord have a dorsal origin (Richardson et al., 2006). opment is tightly regulated by coordinated expression and During gliogenesis stages, early OPCs delaminate from the biochemical interactions of these lineage-specific transcription ventricular zone and subsequently undergo extensive migration to factors. In the developing spinal cord, early oligodendrocyte populate all regions of the spinal cord. Regardless of their origins, precursor cells (OPCs or OLPs) are induced from the Olig1/2+ OPC cells initially express the Olig2 basic helix–loop–helix pMN domain of the ventral neuroepithelium by Sonic hedgehog (bHLH) and retain its expression even after they (Shh)(Trousse et al., 1995; Pringle et al., 1996; Orentas et al., 1999; differentiate into mature (Lu et al., 2000; Lu et al., 2000), from which motor neurons and OPCs are Takebayashi et al., 2000; Zhou et al., 2000). Soon after their birth, OPC cells progressively gain expression of other transcription factors, notably the Sox10 HMG transcriptional regulator and the ⁎ Corresponding author. Fax: +502 852 6228. Nkx2.2 homeodomain transcription factor. In embryonic spinal E-mail address: [email protected] (M. Qiu). cord, expression of Sox10 is selectively up-regulated in OPC cells

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.10.007 684 Z. Liu et al. / Developmental Biology 302 (2007) 683–693 before their emigration into the surrounding regions and persists in Takebayashi et al., 2002; Zhou and Anderson, 2002). mature OLs (Stolt et al., 2002). Nkx2.2 is initially expressed in the However, Olig2 is continuously expressed in differentiating p3 domain and its derived V3 interneurons (Briscoe et al., and mature OPCs, raising the possibility that Olig2 may also 1999; Liu et al., 2003). However, at later stages, Nkx2.2 be involved in OL differentiation and maturation. To expression is specifically up-regulated in Olig2+ OPC cells investigate this possibility, we ectopically expressed Olig2 in either immediately before they migrate out of the ventral embryonic chicken spinal cords by in ovo electroporation and ventricular zone in chicken (Xu et al., 2000; Soula et al., 2001; then examined its effects on myelin gene expression and OL Zhou et al., 2001; Fu et al., 2002) or after they disperse into the differentiation after various time periods. white matter region in rodents (Fu et al., 2002). Following OL In contrast to the previous reports that Olig2 expression differentiation in the white matter, Nkx2.2 expression is alone was not sufficient to induce ectopic oligodendrocyte gradually down-regulated in differentiated oligodendrocytes development (Sun et al., 2001; Zhou et al., 2001), we found (Xu et al., 2000). that Olig2 expression was able to induce expression of the Recent molecular and genetic studies demonstrated that Olig2, early OPC marker Sox10 4 days after electroporation (at Sox10 and Nkx2.2 play distinct roles in OL lineage progression. It cE6) (Figs. 1A–B). However, ectopic expression of Nkx2.2, has been shown that the Olig2 transcription factor is a primary myelin basic protein (MBP) and myelin-associated glyco- determinant of oligodendrocyte fate specification. In Olig2 null protein (MAG) was not observed at this stage (Figs. 1C–D; mutants, OPC generation was completely abolished in the spinal data not shown). Intriguingly, following 1 more day of cord (Lu et al., 2002; Takebayashi et al., 2002; Zhou and Olig2 expression (at cE7), ectopic expression of Nkx2.2 and Anderson, 2002). However, expression of Olig2 alone in myelin genes MBP, and MAG was then detected in the embryonic chicken spinal cord did not promote OPC generation, electroporated side of the cord (Figs. 1E–J). Moreover, and co-expression of Nkx2.2 was required to promote ectopic nearly all the induced MAG+ oligodendrocytes co-expressed formation of OPC cells and precocious oligodendrocyte differ- Olig2 (Figs. 1I, J), suggesting a cell autonomous mechanism entiation (Sun et al., 2001; Zhou et al., 2001). The Nkx2.2 for Olig2 induction of oligodendrocyte differentiation. homeodomain transcription factor plays a crucial role in the Therefore, Olig2 expression alone in the developing spinal terminal differentiation of oligodendrocytes. Null mutation of cord is capable of inducing both the early specification and Nkx2.2 resulted in a dramatic delay and reduction of oligoden- the maturation of OLs. drocyte differentiation although the initial generation of Olig2+ OPCs in the mutants was not compromised (Qi et al., 2001). Expression of Sox10 is sufficient for inducing precocious It has been recently shown that Sox10 also possesses important oligodendrocyte differentiation and ectopic expression of Olig2 functions in promoting terminal differentiation of oligodendro- and Nkx2.2 cytes. In the absence of Sox10 activity, oligodendrocyte differentiation in the spinal cord was dramatically inhibited or The induction of mature OL markers by Olig2 closely delayed (Stolt et al., 2002; Lu et al., 2002). Conversely, over- resembled the ectopic expression of Sox10 but not of Nkx2.2 expression of Sox10 protein in heterologous cells lines can induce (Fig. 1) implies that Sox10 is directly mediating the effects of gene expression from the myelin basic protein (MBP) promoter Olig2 on OL differentiation. To test this possibility, we next (Stolt et al., 2002). However, it is not known whether the in vivo examined the effects of Sox10 over-expression on OL expression of Sox10 has similar effects in the developing CNS. development in embryonic chicken spinal cord. Starting at Although the roles of Olig2, Sox10 and Nkx2.2 in 3 days following Sox10 electroporation (at cE5), induction of oligodendrocyte development have been extensively investi- late differentiation markers MAG, MBP and PLP was gated, the epistatic relationship and functional interactions of observed in the electroporated side of the spinal cords (Figs. these three transcription factors remain to be determined. In the 2A–E). The induction of myelin gene expression by Sox10 present study, we investigated their functional relationship and occurred about 2 days earlier than that induced by Olig2. presented the first in vivo evidence for the reciprocal regulation Surprisingly, Sox10 also induced the expression of early OPC of Olig2 and Sox10 expression in their induction of OL fate and markers Olig2 and Nkx2.2. Double labeling of MAG with differentiation. Sox10 and Nkx2.2 function in parallel and Olig2 or Nkx2.2 revealed that some of the ectopically induced downstream of Olig2 to regulate OL maturation. The control of MAG+ oligodendrocytes did not co-express Olig2 (Fig. 2D) or OL differentiation by Olig2, Sox10 and Nkx2.2 is a dosage- Nkx2.2 (Fig. 2E), suggesting that Sox10 can directly induce dependent developmental process, as suggested by the sig- myelin gene expression in the absence of Olig2 and Nkx2.2 nificant delay of OL maturation in all three heterozygous mice. expression. Moreover, Olig2 and Nkx2.2 were induced by Sox10 over-expression in distinct populations of cells (Figs. Results 2G–H). The ectopic induction of Olig2+ and Nkx2.2+ cells could be further confirmed by the dorsal restricted expression Expression of Olig2 is sufficient for inducing Sox10 and Nkx2.2 of Sox10 (Fig. 2I–J; data not shown), ruling out the possibility expression and precocious oligodendrocyte differentiation that the ectopic Olig2+ or Nkx2.2+ cells resulted from the Sox10-induced dorsal migration of ventrally derived OPCs. Previous studies showed that Olig2 is primarily involved in Together, there results suggested that MAG, Olig2 and Nkx2.2 the early fate specification of OLs (Lu et al., 2002; can be induced by Sox10 independently of each other. .Lue l eeomna ilg 0 20)683 (2007) 302 Biology Developmental / al. et Liu Z. – 693

Fig. 1. Induction of oligodendrocyte cells by Olig2 expression in embryonic chicken spinal cords. Stage 12–13 chicken embryos were electroporated with RCABSP–Olig2 expression vector and allowed to develop further for 4 days (A–D) or 5 days (E–J). Spinal cord sections were then subjected to in situ hybridization with Sox10 (B, F) or MBP (D, H) riboprobes, or subjected to immunofluorescent staining with anti-Olig2 (A, E), anti-Nkx2.2 (C, G), or anti-Olig2 and anti-MAG simultaneously followed by laser confocal scanning (I, J). Panel J is a higher magnification of the outlined area in panel I. Induction of Nkx2.2 by Olig2 in the white matter is represented by arrows in panel G. 685 686 Z. Liu et al. / Developmental Biology 302 (2007) 683–693

Fig. 2. Induction of both early and late oligodendrocyte markers by ectopic expression of Sox10 for 3 days in embryonic chicken spinal cords. (A–F) Induction of oligodendrocyte markers after Sox10 expression in one experiment. Note that many MAG+ cells in the dorsal spinal cord did not co-express Olig2 or Nkx2.2 (D, E). (G, H) Independent induction of Olig2 and Nkx2.2 by Sox10 over-expression. Sox10 transgene expression was detected by . (I, J) Induction of Olig2 by dorsal restricted expression of Sox10.

Oligodendrocyte differentiation is dependent on the gene expression of MBP and PLP was observed in the white matter of dosage of Olig2, Sox10 and Nkx2.2 transcription factors wild-type (Fig. 4A) but not Sox10 and Nkx2.2 homozygous mutant tissues (Figs. 4E–F). As in Olig2+/− embryos, the The induction of OL differentiation by a prolonged activation number of MBP+ /PLP+ mature OLs in these two heterozygotes of Olig2 pathway in chicken embryos implicates that OL was significantly smaller than that in the wild-type tissues (Figs. development may be sensitive to the dosage or level of Olig2 4B, C, G). The reduction of MBP expression was previously expression in the developing spinal cord. To investigate this noticed in Sox10+/− embryos (Stolt et al., 2004). In addition, in possibility, we first compared the expression of mature OL the Sox10+/− and Nkx2.2+/− double heterozygous animals, the markers in E18.5 mouse spinal cords with different dosages of number of MBP+ /PLP+ oligodendrocytes was further Olig2 expression. At this stage, many MBP+ and PLP+ decreased, suggesting that Sox10 and Nkx2.2 mutations have oligodendrocytes were found in the white matter region of the an additive or synergistic effects on OL differentiation. wild-type embryos, but none in the Olig2 homozygous mutants (Figs. 3A–C; Lu et al., 2002, Takebayashi et al., 2002). In the Sox10 mutation leads to a reduced number of Olig2+ and heterozygous animals, the number of MBP+ /PLP+ mature OLs Nkx2.2+ OPCs in the spinal cord was significantly reduced compared to that in the wild-type tissues (Figs. 3A, B, M). The number of Olig2+, Sox10+ and Nkx2.2+ The observation that Sox10 over-expression resulted in an OPCs was similarly reduced in the heterozygous embryos (Figs. increase of Olig2+ cells and later Nkx2.2+ cells (Fig. 2) raises 3D–M). The reduction in the expressions of Olig2, MBP, PLP the possibility that Sox10 may regulate the expression of Olig2 and Sox10 in heterozygous embryos was further confirmed by RT- and Nkx2.2. To investigate this possibility, we first examined the PCR (Figs. 3N, O). The decrease in the number of OPCs and OLs expression of Olig2 in Sox10 mutant spinal cords. At the onset of in Olig2+/− embryos did not appear to result from an increased oligodendrogenesis (E12.5), Olig2+ OPCs started to migrate out apoptosis, as cell death detected by Caspase 3 immunostaining of the ventricular zone (VZ) in all three genotypes. However, the (Figs. S2A–C) or TUNEL staining (data not shown) was similar in number of Olig2+ cells in the ventral ventricular zone (pMN all three genotypes. These observations demonstrated that the domain) and the mantle zone was slightly decreased in both Olig2 activation of OL differentiation in the spinal cord is Sox10 heterozygous and homozygous tissues (Figs. 5A–C, J). dependent on the gene dosage of Olig2. BrdU pulse for two hours revealed a similar cycling rate of The observation that Olig2 controls the expression of Sox10 Olig2+ cells in all three genotypes (Figs. 5A–C, K). The reduc- and Nkx2.2 in oligodendrocyte cells (Figs. 1 and 3) raises the tion of Olig2+ OPCs in the mutant tissues remained apparent at possibility that regulation of oligodendrocyte differentiation by E13.5 (Fig. S1) and E18.5 (Figs. 5D–F, L). The decrease in the these two transcription factors could also be a dosage-dependent number of OPCs in Sox10 mutant embryos could not be account- developmental process. To investigate this possibility, we ed for by differential survival, as a similar apoptosis rate was systematically studied the expression of myelin genes in observed in all three genotypes (Figs. S2D–F). Together, these E18.5 spinal cords of Sox10+/− and Nkx2.2+/− animals (Stolt results suggested that Sox10 may play a role in up-regulating or et al., 2002; Qi et al., 2001; Lu et al., 2002). At this stage, sustaining Olig2 gene expression in oligodendrocyte cells. Z. Liu et al. / Developmental Biology 302 (2007) 683–693 687

Fig. 3. Delayed oligodendrocyte differentiation in Olig2 heterozygous animals. (A–L) Transverse spinal cord sections at the thoracic level from E18.5 wild-type (A, D, G, J), Olig2+/− (B, E, H, K) and Olig2−/− (C, F, I, L) embryos were subjected to in situ hybridization with MBP (A–C) riboprobe, or immunostaining with anti-Olig2 (D–F) and anti-Sox10 (G–I) and anti-Nkx2.2 (J–L). Expression of these markers was significantly reduced in the heterozygous tissues compared to that in the wild- type animals. (M) Statistical analyses of MBP+ cells, Sox10+ cells, and Nkx2.2+ cells in the white matter (those in the gray matter likely represent V3 interneurons) in Olig2 heterozygous embryos relative to wild-type embryos. (N) Semi-quantitative RT-PCR from two different groups of wild-type and heterozygous P0 spinal cord tissues. (O) Quantitation of RT-PCR products in Olig2+/− tissues relative to the wild-type tissues.

To study the effect of Sox10 mutation on Nkx2.2 up- al., 2001), whereas the Nkx2.2-immunoreactive cells in the gray regulation in Olig2+ OPCs, we examined the expression and matter likely represented V3 interneurons (Liu et al., 2003). A distribution of Nkx2.2+ OPCs in E18.5 spinal cord. At this smaller number of Nkx2.2+ differentiating OPCs was observed stage, Nkx2.2+ differentiating OPCs were predominantly in the white matter of Sox10+/− spinal cord and a further located in the white matter region of wild-type tissue (Qi et reduction was detected in the homozygous mutants (Figs. 5G–I, 688 Z. Liu et al. / Developmental Biology 302 (2007) 683–693

Fig. 4. Delayed oligodendrocyte differentiation in Sox10+/− and Nkx2.2+/− heterozygous animals. (A–F) Transverse spinal cord sections at the thoracic level from E18.5 various mutant embryos were subjected to in situ hybridization with MBP riboprobe. (G) Statistical analyses of MBP+ and PLP+ oligodendrocytes in Sox10 and Nkx2.2 heterozygous and homozygous embryos. The number of MBP+ and PLP+ cells was significantly reduced in single heterozygous animals, and further reduced in Sox10+/−; Nkx2.2+/− double heterozygotes.

L). Double labeling experiments revealed that the percentage of Nkx2.2 mutation (Figs. 6D–F), indicating that Nkx2.2 does not Olig2+ cells that co-expressed Nkx2.2 was markedly reduced regulate Sox10 expression in Olig2+ OPCs. Since myelin gene (Figs. 6A–C), suggesting that Sox10 also modulates Nkx2.2 expression was greatly inhibited in Nkx2.2 mutant spinal cords expression in undifferentiated Olig2+ OPC cells. at this stage (Fig. 4F), it also suggested that during normal development, expression of Olig2 and Sox10 in Nkx2.2 mutants Nkx2.2 mutation did not affect Olig2 and Sox10 expression in is not sufficient for driving OL differentiation and the Nkx2.2 OPC cells activity is additionally required to fully achieve the differentia- tion process. We next investigated whether Nkx2.2 activity can regulate Olig2 and Sox10 expression at later stages of OL development. Discussion At E18.5, a slightly larger number of Sox10+ and Olig2+ OPC cells were observed in Nkx2.2 mutant spinal cord (Figs. 6D–F), In this study, we presented the first in vivo evidence that likely due to an expanded pMN domain and subsequently ectopic expression of Olig2 or Sox10 alone was sufficient for increased production of OPCs at earlier stages (Qi et al., 2001). inducing myelin gene expression in the developing spinal cord. Double immunostaining revealed that the percentage of Olig2+ Sox10 and Nkx2.2 appear to function in parallel to regulate OL cells that co-expressed Sox10 was not significantly altered by differentiation process. In addition, we demonstrated that Z. Liu et al. / Developmental Biology 302 (2007) 683–693 689

Fig. 5. Reduced expression of Olig2 and Nkx2.2 in Sox10 mutant embryos. (A–C) E12.5 wild-type (A), Sox10+/− (B) and Sox10−/− (C) embryos were pulse- labeled with BrdU for 2 h and transverse spinal cord sections were subjected to double immunolabeling with anti-BrdU (green) and anti-Olig2 (red). (D–I) Spinal cord sections from E18.5 wild-type (D, G), Sox10+/− (E, H) and Sox10−/− (F, I) embryos were subjected to immunostaining with anti-Olig2 (D–F) or anti-Nkx2.2 (G–I). Expression of these markers was significantly reduced in the heterozygous tissues compared to that in the wild-type animals. (J) Number of Olig2+ cells in E12.5 Sox10+/− and Sox10−/− embryos relative to the wild-type embryos. (K) Percentage of Olig2+ cells that incorporated BrdU at E12.5 in all three genotypes. (J) Number of Olig2+ and Nkx2.2+ cells in E18.5 Sox10+/− and Sox10−/− embryos relative to the wild-type embryos. transcriptional control of OL differentiation by Olig2, Sox10 alone was not sufficient for induction of OL development, and and Nkx2.2 is a dosage-dependent developmental process. co-expression of Nkx2.2 was required for terminal differentia- tion of OLs (Sun et al., 2001; Zhou et al., 2001). In the present Sox10 mediates the Olig2 induction of oligodendrocyte study, we demonstrated that expression of Olig2 alone for differentiation and myelin gene expression 4 days was able to induce the early OPC marker Sox10, and a slightly prolonged expression of Olig2 (for 5 days) can further Olig2 plays an essential role in the fate specification of OL induce expression of the late OPC marker Nkx2.2 and mature lineage, and null mutation of Olig2 resulted in a complete loss markers MAG, MBP and PLP (Fig. 1). The discrepancy of cells of the oligodendrocyte lineage in the spinal cord (Lu et between the present and previous over-expression studies could al., 2002; Takebayashi et al., 2002; Zhou and Anderson, 2002). be accounted for by both the expression level and duration of However, it was also demonstrated that expression of Olig2 Olig2 expression. In our studies, we employed a higher 690 Z. Liu et al. / Developmental Biology 302 (2007) 683–693

Fig. 6. Cross-regulation of Olig2, Sox10 and Nkx2.2 expression in oligodendrocyte cells. (A–C) Sox10 mutation affects the expression of Nkx2.2 in Olig2+ OPCs. E18.5 spinal cord sections from wild-type (A) and Sox10−/− (B) embryos were simultaneously immunostained with antibodies against Olig2 and Nkx2.2, and the percentage of Olig2+ cells that co-express Nkx2.2 in wild-type and Sox10 mutants was calculated (C). (D–F) Nkx2.2 mutation does not reduce the expression of Olig2 and Sox10. E18.5 spinal cord sections from wild-type (D) and Nkx2.2−/− (E) embryos were simultaneously immunostained with antibodies against Olig2 and Sox10, and the percentage of Olig2+ cells that co-expressed Sox10 in wild-type and Nkx2.2 mutants was calculated (F). concentration (2 μg/μl) of RCASBP retroviral expression 2005). Thus, Sox10 functions to promote glial genesis and vectors to achieve a robust and sustained expression of Olig2 suppress neurogenesis during neural development. At early for a longer time period. Ectopic expression of mature OL stages, Sox10 is expressed in the dorsal tips and promotes the markers was difficult to detect in embryos electroporated with formation and migration of neural crest precursor cells, 1 μg/μl or lower concentration of Olig2 expression vector (data whereas at later stages, it is expressed in OPCs (Stolt et al., not shown), consistent with the concept of dosage-dependent 2002) and promotes the terminal differentiation of oligoden- activation of OL differentiation by oligodendrocyte lineage drocytes in the CNS. Ectopic and sustained expression of transcription factors (as discussed below). Sox10 in young embryonic spinal cord appeared to recapitulate Sox10 appears to directly mediate the effects of Olig2 on both functions. oligodendrocyte differentiation based on the present and some previous studies. Firstly, in Olig2-transfected spinal cord, the Sox10 and Nkx2.2 function in parallel to control pattern of induced MBP and PLP expression closely resembled oligodendrocyte differentiation that of ectopic Sox10 expression (Fig. 1). Secondly, expression of Sox10 alone in chicken spinal cord was able to induce Sox10 and Nkx2.2 are both required for OL differentiation expression of myelin genes MAG, MBP and PLP in a much and maturation, and mutations in these two transcription factors shortened time frame (Fig. 2), as compared to the Olig2 exhibit similar phenotypes with delayed and retarded OL induction of myelin gene expression. Thirdly, loss of Sox10 differentiation (Qi et al., 2001; Stolt et al., 2002). In their activityresultedinaseverereductionofmyelingene heterozygous embryos, the differentiation of oligodendrocytes expression and OL differentiation (Stolt et al., 2002). In is significantly decreased and delayed. Interestingly, there is light of the findings that Sox10 is the only transcription factor a further reduction of oligodendrocyte differentiation in the that is known to be expressed in myelin-forming cells in both Sox10+/− and Nkx2.2+/− double heterozygous animals (Fig. CNS and PNS and its mutation affects myelin gene expression 4), suggesting that Nkx2.2 and Sox10 have synergistic effects in both tissues (Wegner and Stolt, 2005), it is possible that on OL differentiation. Sox10 may be directly responsible for stimulating myelin- Despite the observations that Sox10 over-expression can specific gene expression and cell differentiation in oligoden- directly stimulate myelin gene expression (Fig. 2; Stolt et al., drocytes and Schwann cells. Previous studies demonstrated 2002; Wei et al., 2004), normal expression of Sox10 in OPCs that over-expression of Sox10 and its related SoxE member did not appear to be sufficient for OL differentiation in vivo.In Sox9 also induced neural crest-like phenotype at the expense E18.5 Nkx2.2 null mutants, expression of both Sox10 and Olig2 of neurogenesis (Cheung and Briscoe, 2003; McKeown et al., was not compromised (Fig. 6) but OL differentiation was Z. Liu et al. / Developmental Biology 302 (2007) 683–693 691 severely retarded (Qi et al., 2001), implying that during normal of Sox10 gene resulted in a decreased expression of Olig2 in the CNS development, Sox10 activation of MBP/PLP genes is spinal cord (Figs. 5 and 6). The small reduction of Olig2+ OPCs negatively regulated by an unknown inhibitory molecule. Given was previously unnoticed by in situ RNA hybridization (Stolt et that Nkx2.2 primarily functions as a transcriptional repressor al., 2002) possibly due to its lower detection sensitivity. The during neural development (Muhr et al., 2001; Zhou et al., positive feedback on Olig2 expression by Sox10 also implies 2001; Wei et al., 2005), it is possible that Nkx2.2 may regulate that Sox10 may have an important function in up-regulating and/ the terminal differentiation of oligodendrocytes in an indirect or maintaining Olig2 expression during oligodendrocyte manner. Conceivably, Nkx2.2 may repress the expression or development. activity of an inhibitory factor that prevents OL differentiation Similarly, Sox10 expression can also regulate Nkx2.2 and myelin gene expression. expression, and this regulatory relationship may contribute to Taken into account all these observations, we propose that the up-regulation of Nkx2.2 protein in differentiating OPCs oligodendrocyte differentiation is controlled by two parallel (Fig. 7). In the absence of Sox10 function, Nkx2.2 expression is pathways, a positive regulatory pathway mediated through dramatically down-regulated but not completely inhibited (Fig. Sox10 and an inhibitory pathway mediated by an unknown 6). Conversely, over-expression of Sox10 can promote Nkx2.2 factor whose expression or activity can be repressed by Nkx2.2 expression in embryonic spinal cord independently of Olig2 (Fig. 7). While Sox10 may directly stimulate gene expression induction (Fig. 2), suggesting that the induction of Nkx2.2 and from myelin gene promoters, Nkx2.2 might repress the Olig2 by Sox10 is mediated by distinct molecular pathways or expression or activity of the inhibitory factor. It is conceivable mechanisms. that in OPC cells, this hypothetical inhibitory factor can bind to Sox10 protein and therefore interfere with Sox10 activity. Dosage-dependent regulation of oligodendrocyte development Nkx2.2 may repress its expression as a transcription repressor or by transcriptional factors its activity by physical association with this inhibitory factor. In this regard, Nkx2.2 protein may facilitate Olig2- and Sox10- The haploinsufficiency in OL differentiation was previously mediated OL differentiation and function to control the timing reported for the Sox10 transcriptional regulator (Stolt et al., of OL differentiation. However, a constitutive high level of 2004). Here we showed that the delayed maturation of OLs was Sox10 or Olig2 expression can obviate the requirement of also observed in Olig2 and Nkx2.2 heterozygous animals, Nkx2.2 in OL differentiation and lead to ectopic myelin gene indicating that the OL differentiation process is particularly expression in the absence of Nkx2.2 activity (Fig. 2). sensitive to the gene dosage of these transcriptional factors. The dosage-dependent OL development could also explain the Regulation of Olig2 and Nkx2.2 expression by Sox10 in OPC delayed OL differentiation phenotype in other mutants (e.g. cells Nkx6.1−/− and Gli2−/− mutants) in which the Olig2 expression in the pMN domain was markedly reduced at the stage of Although genetic studies have established that Sox10 gliogenesis (Liu et al., 2003; Qi et al., 2003). functions downstream of Olig2 (Lu et al., 2002; Takebayashi The developmental defects caused by haploinsufficiency et al., 2002; Zhou and Anderson, 2002), both gain- and loss-of- have recently been noticed for a number of other transcription function studies suggested that there is a reciprocal regulation of factors in a diversity of developmental processes (Aoki et al., Olig2 and Sox10 expression in oligodendrocyte cells. In 2004; Bland et al., 2000; Kalinichenko et al., 2004). For embryonic chicken spinal cord, over-expression of Sox10 can instance, a decrease in Sox9 dosage by haploinsufficiency in weakly induce Olig2 expression (Fig. 2). Conversely, mutation neural crest-derived tissues resulted in delayed frontal cranial suture closure (Sahar et al., 2005). Mice heterozygous for Pax6 mutations display small eyes (Sey) phenotype with a small lens (van Raamsdonk and Tilghman, 2000). Although many transcription factor genes display semi-dominant loss-of- function heterozygous phenotypes, the underlying mechanism for this dosage requirement is not known. One plausible explanation is that a critical threshold of transcription factors is required for cell differentiation and that the time in development for reaching this threshold level is delayed in heterozygotes. Consistent with this hypothesis, oligodendrocyte differentiation and maturation is simply delayed in animals heterozygous for Olig2, Sox10 and Nkx2.2. However, the initial delay of OL differentiation in these heterozygous animals is at least in part compensated during further development, given the more or less normal oligodendrocyte phenotype in adult heterozygous Fig. 7. A hypothetical hierarchy and functional interaction of transcription mice. In further support of the threshold theory, a strong or factors that control oligodendrocyte development. Dashes represent weak prolonged activation of the Olig2 or Sox10 pathway is capable regulations. of promoting ectopic and precocious OL differentiation and 692 Z. Liu et al. / Developmental Biology 302 (2007) 683–693 myelin gene expression (Figs. 1 and 2). Therefore, OL Acknowledgments differentiation appears to be exquisitely sensitive to the dosage of Olig2/Sox10 gene expression and can be affected by both We are very grateful to Drs. Charles Stiles, David Rowitch haploinsufficiency and overdosage. A similar mechanism may and Richard Lu for generously providing the Olig1/Olig2 mutant operate in the control of skeletal growth by the SHOX mouse lines and the anti-Olig2 antibody and for their critical homeodomain transcription factor in human development reading of the manuscript. This work was supported by NIH (Ogata et al., 2001). (NS37717) and by National Multiple Sclerosis Society (RG 3275). Experimental procedures Appendix A. Supplementary data Genotyping of Olig2, Sox10 and Nkx2.2 mutant mice Supplementary data associated with this article can be found, The Olig2, Sox10 and Nkx2.2 homozygous null embryos were obtained by in the online version, at doi:10.1016/j.ydbio.2006.10.007. the interbreeding of double heterozygous animals. Genomic DNA extracted from embryonic tissues or mouse tails was used for genotyping by Southern analysis or by PCR. Genotyping of Olig2, Sox10 and Nkx2.2 loci was described References earlier (Lu et al., 2002; Stolt et al., 2002; Qi et al., 2001). Aoki, Y., Mori, S., Kitajima, K., Yokoyama, O., Kanamaru, H., Okada, K., In situ RNA hybridization and immunofluorescent staining Yokota, Y., 2004. Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans. Genes Cells 9, 1287–1296. Bland, M.L., Jamieson, C.A., Akana, S.F., Bornstein, S.R., Eisenhofer, G., Spinal cord tissues at the thoracic level were isolated from E10.5 to E18.5 Dallman, M.F., Ingraham, H.A., 2000. Haploinsufficiency of steroidogenic mouse embryos and then fixed in 4% paraformaldehyde at 4°C overnight. factor-1 in mice disrupts adrenal development leading to an impaired stress Following fixation, tissues were transferred to 20% sucrose in PBS overnight, response. Proc. Natl. Acad. Sci. U. S. A. 97, 14488–14493. embedded in OCT media and then sectioned (20 μm thickness) on a cryostat. Briscoe, J., Sussel, L., Serup, P., Hartigan-O'Conner, D., Jessell, T.M., Adjacent sections from the wild-type and mutant embryos were subsequently Rubenstein, J.L., Ericson, J., 1999. gene Nkx-2.2 and subjected to in situ hybridization (ISH) or immunofluorescent staining. ISH specification of neuronal identity by graded sonic hedgehog signaling. was performed as described in Schaeren-Wiemers and Gerfin-Moser (1993) Nature 398, 622–627. with minor modifications, and the detailed protocol is available upon request. Cai, J., Qi, Y., Hu, X., Tan, M., Liu, Z., Zhang, J., Li, Q., Sander, M., Qiu, M., Double immunofluorescent procedures were previously described in Xu et al. 2005. Generation of oligodendrocyte precursor cells from mouse dorsal (2000). The dilution ratio of antibodies is as follows: anti-Olig2 (1:6000), anti- spinal cord independent of Nkx6-regulation and Shh signaling. Neuron 45, MAG (Chemicon, 1:500), anti-Myc (Sigma, 1:50), anti-Nkx2.2 (DSHB, 1:50), 41–53. anti-Sox10 (1:3000; Stolt et al., 2003) and anti-caspase 3 (Cell Signaling, Cheung, M., Briscoe, J., 2003. Neural crest development is regulated by the 1:400). transcription factor Sox9. Development 130, 5681–5693. Fogarty, M., Richardson, W., Kessaris, N., 2005. A subset of oligodendrocytes Semi-quantitative RT-PCR generated from radial glia in the dorsal spinal cord. Development 132, 1951–1959. − Total RNA was extracted from spinal cord of P0 wt and Olig2+/ mice by Fu, H., Qi, Y., Tan, M., Cai, J., Hirohide, T., Nakafuku, M., Richardson, W., Qiu, using Trizol (Invitrogen). The concentration of total RNA was assessed by M., 2002. Dual origin of spinal oligodendrocyte progenitors and evidence spectrophotometry and adjusted roughly to the same concentrations among for the cooperative role of Olig2 and Nkx2.2 in the control of samples. First-strand cDNA was generated by using SuperScript III first-strand oligodendrocyte differentiation. Development 129, 681–693. synthesis system (Invitrogen). To ensure that PCR cycles ended before Kalinichenko, V., Gusarova, G., Kim, I., Shin, B., Yoder, H., Clark, J., saturation, generally, the cycle number for each primer was first tried at 27 Sapozhnikov, A., Whitsett, J., Costa, R., 2004. Foxf1 haploinsufficiency – cycles and then decreased or increased 2 5 cycles, depending on the intensity of reduces Notch-2 signaling during mouse lung development. Am. J. Physiol.: the initial PCR products. Therefore, each group of samples were run for PCR Lung Cell Mol. Physiol. 286, L521–L530. with at least two or three different cycle numbers to select the reaction showing Liu, R., Cai, J., Hu, X., Tan, M., Qi, Y., German, M., Rubenstein, J., Sander, M., clear bands with the lowest cycle number. Three different groups of samples Qiu, M., 2003. Region-specific and stage-dependent regulation of Olig gene were used. Primers for a housekeeping gene (GAPDH) were used as a control. expression and oligodendrogenesis by Nkx6.1 homeodomain transcription ′ Primer sequences used for analyses are as follows: GAPDH, forward 5 - factor. Development 130, 6221–6231. ′ ′ ′ ′ ggccttccgtgttcctac-3 , reverse 5 -ctgttgctgtagccgtattca-3 ; MBP, forward 5 - Lu, Q., Yuk, D., Alberta, J., Zhu, Z., Pawlitsky, I., Chan, J., McMahon, A., ′ ′ ′ ccctcagagtccgacgagctt-3 , reverse 5 -tcccttgtgagccgatttat-3 ; Nkx2.2, forward Stiles, C., Rowitch, D., 2000. Sonic Hedgehog-regulated oligodendrocyte ′ ′ ′ ′ 5 -gaccttccggacaccaacgat-3 , reverse 5 -gcgagggcagaggcgtcac-3 ; Olig2, for- lineage genes encoding bHLH proteins in the mammalian central nervous ′ ′ ′ ′ ward 5 -ccgaaaggtgtggatgcttat-3 , reverse 5 -tcgctcaccagtcgcttcat-3 ;PLP, system. Neuron 25, 317–329. ′ ′ ′ ′ forward 5 -gacatgaagctctcactggtac-3 , reverse 5 -catacattctggcatcagcgc-3 ; Lu, Q., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C., Rowitch, D., 2002. ′ ′ ′ ′ Sox10, forward 5 -aagagcaagccgcacgtc-3 , reverse 5 -acgttgccgaagtcgatgtgg-3 . Common developmental requirement for Olig function indicates a /oligodendrocyte connection. Cell 109, 75–86. In ovo electroporation McKeown, S.J., Lee, V.M., Bronner-Fraser, M., Newgreen, D.F., Farlie, P.G., 2005. Sox10 overexpression induces neural crest-like cells from all Full-lengths mouse Olig2, Sox10 and Nkx2.2 cDNAs were subcloned into dorsoventral levels of the neural tube but inhibits differentiation. Dev. the replication-competent retroviral vector RCASBP(B) (Morgan and Fekete, Dyn. 233, 430–444. 1996). For in ovo electroporation, about 1.5 μl(2μg/μl) of expression vectors Miller, R.H, 2002. Regulation of oligodendrocyte development in the vertebrate was injected into stage 12–13 white horn chicken embryos with the aid of CNS. Prog. Neurobiol. 67, 451–467. Picospritzer III instrument. The injected embryos were then subjected to three Morgan, B., Fekete, D., 1996. Manipulating gene expression with replication- short-pulses of electrical shock (25 V, 50 ms for each pulse) and allowed to competent retroviruses. In: Bronner-Fraser, M.E. (Ed.), Methods in Avian develop for various time periods before they were fixed in 4% PFA for gene Embryology. Academic Press, San Diego, pp. 185–218. expression studies. Muhr, J., Andersson, E., Persson, M., Jessell, T., Ericson, J., 2001. Z. Liu et al. / Developmental Biology 302 (2007) 683–693 693

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