Terminal Differentiation Of Oligodendrocytes Research Articles

Differential activity of transcription factor Sox9 in early and adult oligodendroglial progenitor cells.

The high-mobility-group domain-containing transcription factor Sox9 confers glial competence to neuroepithelial precursors in the developing central nervous system and is an important determinant of astroglial and oligodendroglial specification. In oligodendroglial cells, it remains expressed in oligodendrocyte progenitor cells (OPCs) of the developing nervous system, but is shut off in differentiating oligodendrocytes as well as in OPCs that persist in the adult nervous system. To better understand the role of Sox9 in OPCs, we generated mouse models that allowed oligodendroglial expression of a Sox9 transgene during development or in the adult. With transgene expression beginning in the last trimester of pregnancy, the number of OPCs increased dramatically, followed by comparable gains in the number of pre-myelinating and myelinating oligodendrocytes as assessed by marker gene expression. This argues that Sox9 boosts oligodendrogenesis during ontogenetic development at all stages, including terminal oligodendrocyte differentiation. When Sox9 transgene expression started in the adult, many transgene-expressing OPCs failed to maintain their progenitor cell identity and instead converted into myelinating oligodendrocytes. As infrequent and inefficient differentiation of adult OPCs is one of the main obstacles to effective remyelination in demyelinating diseases such as Multiple Sclerosis, increased Sox9 levels in adult OPCs may substantially increase their remyelination capacity.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

Differential Inhibition of Sox10 Functions by Notch-Hes Pathway.

In the developing central nervous system, the terminal differentiation of oligodendrocytes (OLs) is regulated by both extrinsic and intrinsic factors. Recent studies have suggested that the Notch-Hes signaling pathway influences the maturation of oligodendrocytes in culture and during development. However, the specific Notch receptors and their downstream effectors Hes genes that are involved in oligodendrocyte maturation have not been investigated systematically. In this study, we showed that Notch1 and Notch3 are expressed in oligodendrocyte precursor cells (OPCs) during gliogenesis, and Hes5 is the major Notch downstream transcription factor that is transiently expressed in OPCs. Overexpression of Notch intracellular domain (NICD) and Hes5 proteins in embryonic chicken spinal cord suppressed both the endogenous and Sox10-induced Mbp gene expression. Unexpectedly, overexpression of NICD/Hes5 did not inhibit Sox10 induction of Olig2 expression and Myrf induced Mbp expression, suggesting the differential inhibitory effects of NICD/Hes5 signaling on Sox10 activation of myelin-related genes and early progenitor genes.

Biological functions of sulfoglycolipids and the EMARS method for identification of co-clustered molecules in the membrane microdomains.

Two major sulfoglycolipids, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol) exist in mammals. Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is identical and synthesized by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). We have purified CST homogenously, cloned the CST gene and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. Analysis of CST-null mice has revealed that sulfatide is an essential component for the axo-glial junction at the paranode region and regulates terminal differentiation of oligodendrocytes, and that seminolipid is responsible for the formation of a functional lactate transporter assembly to take up the critical energy source for spermatocytes. We have developed a new analytical method termed EMARS to identify co-clustered molecules in the membrane microdomains in order to elucidate the functional molecules that collaborate with sulfoglycolipids.

Transcription factor Sox10 regulates oligodendroglial Sox9 levels via microRNAs.

During development of myelin-forming oligodendrocytes in the central nervous system the two closely related transcription factors Sox9 and Sox10 play essential roles that are partly shared and partly unique. Whereas Sox9 primarily functions during oligodendroglial specification, Sox10 is uniquely required to induce terminal differentiation and myelination. During this process, Sox10 protein levels rise substantially. As this coincides with a reciprocal decrease in Sox9, we postulated that Sox10 influences Sox9 amounts in differentiating oligodendrocytes. Here we show that Sox9 levels are indeed inversely coupled to Sox10 levels such that Sox10 deletion in oligodendroglial cells evokes a reciprocal increase in Sox9. We furthermore provide evidence that this coupling involves upregulation of microRNAs miR335 and miR338 as direct transcriptional targets of Sox10. The two microRNAs in turn recognize the 3'-UTR of Sox9 mRNA and may thereby reduce Sox9 protein levels posttranscriptionally in oligodendroglial cells. Such a mechanism may enable oligodendroglial cells to adapt the ratio of both related Sox proteins in a manner required for successful lineage progression and differentiation. Mathematical modeling furthermore shows that the identified regulatory circuit has the potential to convert a transient stimulus into an irreversible switch of cellular properties and may thus contribute to terminal differentiation of oligodendrocytes.

Additive dominant effect of a SOX10 mutation underlies a complex phenotype of PCWH

Distinct classes of SOX10 mutations result in peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease, collectively known as PCWH. Meanwhile, SOX10 haploinsufficiency caused by allelic loss-of-function mutations leads to a milder non-neurological disorder, Waardenburg–Hirschsprung disease. The cellular pathogenesis of more complex PCWH phenotypes in vivo has not been thoroughly understood. To determine the pathogenesis of PCWH, we have established a transgenic mouse model. A known PCWH-causing SOX10 mutation, c.1400del12, was introduced into mouse Sox10-expressing cells by means of bacterial artificial chromosome (BAC) transgenesis. By crossing the multiple transgenic lines, we examined the effects produced by various copy numbers of the mutant transgene. Within the nervous systems, transgenic mice revealed a delay in the incorporation of Schwann cells in the sciatic nerve and the terminal differentiation of oligodendrocytes in the spinal cord. Transgenic mice also showed defects in melanocytes presenting as neurosensory deafness and abnormal skin pigmentation, and a loss of the enteric nervous system. Phenotypes in each lineage were more severe in mice carrying higher copy numbers, suggesting a gene dosage effect for mutant SOX10. By uncoupling the effects of gain-of-function and haploinsufficiency in vivo, we have demonstrated that the effect of a PCWH-causing SOX10 mutation is solely pathogenic in each SOX10-expressing cellular lineage in a dosage-dependent manner. In both the peripheral and central nervous systems, the primary consequence of SOX10 mutations is hypomyelination. The complex neurological phenotypes in PCWH patients likely result from a combination of haploinsufficiency and additive dominant effect.

Role of endogenous psychosine accumulation in oligodendrocyte differentiation and survival: Implication for Krabbe disease

Krabbe disease is a lethal, demyelinating condition caused by genetic deficiency of galactocerebrosidase (GALC) and resultant accumulation of its cytotoxic substrate, psychosine (galactosylsphingosine), primarily in oligodendrocytes (OLs). Psychosine is generated by galactosylation of sphingosine by UDP-galactose:ceramide galactosyltransferase (CGT), a galactosylceramide synthesizing enzyme which is primarily expressed in OLs. The expression of CGT and the synthesis of galactosyl-sphingolipids are associated with the terminal differentiation of OL, but little is known about the participation of endogenous psychosine accumulation in OL differentiation under GALC deficient conditions. In this study, we report that accumulation of endogenous psychosine under GALC deficient Krabbe conditions impedes OL differentiation process both by decreasing the expression of myelin lipids and protein and by inducing the cell death of maturating OLs. The psychosine pathology under GALC deficient conditions involves participation of secretory phospholipase A2 (sPLA2) activation and increase in its metabolites, as evidenced by attenuation of psychosine-induced pathology by treatment with pharmacological inhibitor of sPLA2 7,7-dimethyleicosadienoic acid (DEDA). These observations suggest for potential therapeutic efficacy of sPLA2 inhibitor in Krabbe disease.

Dystroglycan modulates the ability of insulin‐like growth factor‐1 to promote oligodendrocyte differentiation

The adhesion receptor dystroglycan positively regulates terminal differentiation of oligodendrocytes, but the mechanism by which this occurs remains unclear. Using primary oligodendrocyte cultures, we identified and examined a connection between dystroglycan and the ability of insulin-like growth factor-1 (IGF-1) to promote oligodendrocyte differentiation. Consistent with previous reports, treatment with exogenous IGF-1 caused an increase in MBP protein that was preceded by activation of PI3K (AKT) and MAPK (ERK) signaling pathways. The extracellular matrix protein laminin was further shown to potentiate the effect of IGF-1 on oligodendrocyte differentiation. Depletion of the laminin receptor dystroglycan using siRNA, however, blocked the ability of IGF-1 to promote oligodendrocyte differentiation of cells grown on laminin, suggesting a role for dystroglycan in IGF-1-mediated differentiation. Indeed, loss of dystroglycan led to a reduction in the ability of IGF-1 to activate MAPK, but not PI3K, signaling pathways. Pharmacological inhibition of MAPK signaling also prevented IGF-1-induced increases in myelin basic protein (MBP), indicating that MAPK signaling was necessary to drive IGF-1-mediated enhancement of oligodendrocyte differentiation. Using immunoprecipitation, we found that dystroglycan, the adaptor protein Grb2, and insulin receptor substrate-1 (IRS-1), were associated in a protein complex. Taken together, our results suggest that the positive regulatory effect of laminin on oligodendrocyte differentiation may be attributed, at least in part, to dystroglycan's ability to promote IGF-1-induced differentiation.

Haloperidol promotes proliferation but inhibits differentiation in rat oligodendrocyte progenitor cell culturesThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

Haloperidol is a commonly used, typical, antipsychotic drug (APD) that acts strongly against positive symptoms, but has fewer therapeutic effects on, or may even aggravate, negative symptoms and cognitive deficits in patients with schizophrenia. Loss of oligodendrocytes has been suggested as a factor associated with the negative symptoms of schizophrenia. Recent study shows that chronic haloperidol treatment induced down-regulation of oligodendrocyte-related genes in certain brain regions of mouse. In this study, we used primary oligodendrocyte progenitor cell cultures from 1- to 3-day-postnatal rats to investigate the direct effects of haloperidol on the proliferation and differentiation of oligodendrocyte progenitor cells. Our results showed that (i) haloperidol (0-10.0 micromol.L-1) facilitated the proliferation of oligodendrocyte progenitor cells, (ii) chronic haloperidol (0.5 micromol.L-1) treatment decreased the number of myelin basic protein positive oligodendrocytes and reduced the oligodendrocytes cells possessing myelin-like membranes, resulting in inhibition of the terminal differentiation of oligodendrocytes, and (iii) D3 receptor mRNA was detected in oligodendrocyte progenitor cells, and haloperidol treatment induced a down-regulation of D3 receptor mRNA. These results suggest that the typical antipsychotic drug haloperidol affects the development of oligodendrocyte progenitor cells, and that D3 receptor down regulation may be involved. Our observations provide new insight into possible cellular mechanisms responsible for the side effects of typical antipsychotic drugs and support the concept that abnormality of oligodendrocytes may be involved in the pathogenesis of schizophrenia.

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4(+) oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5(+) oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage.

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7±6.9%), using hepatocyte growth factor (HGF) and G5 supplement (containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1×) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation.

Subcortical oligodendrocyte- and astrocyte-associated gene expression in subjects with schizophrenia, major depression and bipolar disorder

Deficits in the expression of oligodendrocyte and myelin genes have been described in numerous cortical regions in schizophrenia and affective disorders; however, relatively little attention has been paid to subcortical structures. Here we employed quantitative real time PCR to examine the mRNA expression of 17 genes that are expressed by oligodendrocyte precursors (OLPs) and their derivatives, including astrocytes. Four subcortical regions were examined (the anteroventral (AV) and mediodorsal thalamic nuclei (MDN), internal capsule (IC) and putamen (Put)) in postmortem material from subjects (age 25–68 at time of death) with no known psychiatric history (NCs) as well as in subjects with schizophrenia (SZ), major depressive disorder (MDD), and bipolar disorder (BPD). In all regions examined, genes expressed after the terminal differentiation of oligodendrocytes tended to have lower levels of mRNA expression in subjects with SZ compared to NCs. These differences were statistically significant across regions for four genes (CNP, GALC, MAG and MOG) and approached significance for TF. No genes were under expressed in MDD. Only TF was under expressed in BPD and only in the IC. In contrast, two astrocyte-associated genes (GFAP and ALDH1L1) had higher mean expression levels across regions in all psychiatric groups relative to NCs. These differences reached statistical significance for SZ and MDD relative to NCs. There were no age by diagnosis interactions. The majority of age regressions had negative slopes for the expression of oligodendrocyte-associated genes. GFAP but not ALDH1L1 expression was significantly and positively correlated with age in the MDN, AV and Put. Across subject groups the expression of both astrocyte genes was highly correlated with cumulative neuroleptic exposure in all regions except the Put. Significant positive correlations were also observed in some regions between cumulative neuroleptic exposure and the expression of genes associated with mature oligodendrocytes as well as with bipotential OLPs. Multiple negative correlations were observed between the mRNA expression of astrocyte genes and genes expressed by terminally differentiated oligodendrocytes. These data are discussed in the context of myelin turnover and potential effects of psychiatric illness as well as medications on the developmental fate of OLPs.

Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression

In neurons, serum response factor (SRF)-directed transcription regulates migration, axon pathfinding and synapse function. We found that forebrain-specific, neuron-restricted SRF ablation in mice elevated oligodendrocyte precursors while inhibiting terminal oligodendrocyte differentiation. Myelin gene and protein expression were downregulated and we observed a lack of oligodendrocytes in mixed neuron/glia and oligodendrocyte-enriched cultures derived from Srf(-/-) mutants. Ultrastructural inspection revealed myelination defects and axonal degeneration in Srf(-/-) mutants. Consistent with our finding that neuronal SRF depletion impaired oligodendrocyte fate in a non-cell autonomous manner, neuron-restricted expression of constitutively active SRF-VP16 affected neighboring oligodendrocyte maturation. Genome-wide transcriptomics identified candidate genes for paracrine regulation of oligodendrocyte development, including connective tissue growth factor (CTGF), whose expression is repressed by SRF. Adenovirus-mediated CTGF expression in vivo revealed that CTGF blocks excessive oligodendrocyte differentiation. In vitro, CTGF-mediated inhibition of oligodendrocyte maturation involved sequestration and thereby counteraction of insulin growth factor 1-stimulated oligodendrocyte differentiation.

Hypomorphic Sox10 alleles reveal novel protein functions and unravel developmental differences in glial lineages

The transcription factor Sox10 regulates early neural crest development, specification of neural crest-derived lineages and terminal differentiation of oligodendrocytes in the central nervous system. Here, we generated two novel hypomorphic Sox10 alleles in the mouse. Mutant mice either expressed a Sox10 protein with a triple alanine substitution in the dimerization domain, or a Sox10 protein with a deletion in the central portion that we define as a cell-specific transactivation domain. Phenotypic analysis revealed important roles for a functional dimerization domain and the newly defined novel transactivation domain in melanocyte and enteric nervous system development, whereas early neural crest development and oligodendrocyte differentiation were surprisingly little disturbed in both mutants. Unique requirements were additionally detected for the novel transactivation domain in satellite glia differentiation and during Schwann cell myelination, whereas DNA-dependent dimerization was needed for immature Schwann cells to enter the promyelinating stage. These two hypomorphic alleles thus uncover novel functions of Sox10 in satellite glia and Schwann cells during late developmental stages and reveal important developmental differences between these two types of peripheral glia and oligodendrocytes regarding their reliance on Sox10.

An animal model for late onset chronic demyelination disease caused by failed terminal differentiation of oligodendrocytes

Various animal models are available for studying human multiple sclerosis (MS). Most of them model the initial phase of MS,including the immune-triggered attack of the myelin membrane and/or oligodendrocytes and, occasionally, demonstrate there mission and relapsing phases. However, few mimic the late chronic demyelinating phase. Overexpression of the proteolipid protein gene (Plp) causes a unique demyelinating disorder in mice in which normal-appearing myelin forms early in life and chronic demyelination occurs later. We found that remyelination is severely affected in this late demyelinating phase, but is not caused by deprivation of oligodendrocyte progenitors expressing PDGF receptor alpha (PDGFRa) and Olig2, which are present at an even higher number in the demyelinated white matter of the mutants than in wild-type controls. Furthermore, mature oligodendrocytes containing PLP were observed, but failed to remyelinate. The ability of oligodendrocytes from older transgenic animals to produce a myelin membrane-like structure was not impaired when cultured in vitro, which indicates that the lack of remyelination is not simply caused by changes in the intrinsic properties of the oligodendrocytes. Glial activation also occurred much earlier than active demyelination in mutant mice. Thus, in addition to intrinsic mechanisms, extrinsic mechanisms might also have an important role in defects of remyelination. These features are also observed in patients at a late stage of MS, leading to chronic demyelinating lesions. Thus, this mouse model partly mimics the late stage of MS and can be used to study the cause of inhibition of remyelination.

Gli3 mutation rescues the generation, but not the differentiation, of oligodendrocytes in Shh mutants

In the developing spinal cord, early oligodendrocyte progenitor cells are induced from the ventral neural tube by the Sonic hedgehog ( Shh) signal. Previous studies suggested that the Gli3 zinc finger transcription factor is an important signal transducer of Shh signaling during animal development . Here, we demonstrated that Gli3 plays a non-essential role in ventral oligodendrogenesis during normal development. However, in the absence of Shh signaling, Gli3 functions as a repressor of ventral oligodendrogenesis and ventral oligodendrogenesis is restored in Shh−/−Gli3−/− mutants. Interestingly, despite a normal production of oligodendrocyte precursor cells in the double mutants, oligodendrocyte differentiation is completely suppressed, implying that Shh signaling plays an important role in the terminal differentiation of oligodendrocytes.

199 DIFFERENTIATION OF HIGHLY ENRICHED OLIGODENDROCYTE PRECURSORS AND MATURE OLIGODENDROCYTES FROM RHESUS MONKEY EMBRYONIC STEM CELLS

Generating homologous oligodendrocytes are required for studying the molecular mechanisms of oligodendrogliogenesis and for providing donor cells for transplantation therapies. Previous studies have shown that embryonic stem (ES) cells can be induced to generate neural stem cells with many kinds of culture systems; however, few or no oligodendrocytes were obtained from these culture systems. Here we present a simple method containing five steps for obtaining highly enriched oligodendrocyte precursors (75 � 6.8%) and mature oligodendrocytes (81 � 8.6%) from rhesus monkey embryonic stem (rES) cells. We expanded rES cells on a feeder layer of irradiated MESF (ear skin fibroblasts from a one-week-old rhesus monkey), formed embryoid bodies (EBs), promoted Day 9 (3 days in hanging drop and 6 days in suspension) differentiation into highly enriched (90.2 � 6.1%) neural progenitors (NPs) with hepatocyte growth factor (HGF) and G5 supplement [containing 5 ng/mL (bFGF) and 10 ng/mL epidermal growth factor (EGF)], purified NPs with 0.0625% trypsin in 0.04% EDTA (98% of cells were nestin-positive), amplified those progenitors in HGF and G5 media for two months, and then induced oligodendrocyte precursors differentiation in the absence of G5, but in the presence of 20 ng/mL HGF for 2 days. To obtain terminal oligodendrocytes, neurospheres cultured for 2 months were plated on laminin-coated plates for 3 weeks in the presence of HGF. The results showed that differentiated cells expressed myelin basic protein (MBP) and had typical mature oligodendrocyte morphology. Our studies also revealed that HGF significantly increased the NP proliferation speed (P < 0.05) by both decreasing cell apoptosis rate (P < 0.05) and shortening cell cycle time (P < 0.05) in the presence of G5. Additionally, HGF promoted oligodendrocyte maturation by increasing the length and number of branches and the expression of MBP. To test whether the original HGF had similar functions for oligodendrocyte specification, a series of experiments were evaluated by adding HGF or G5 to differentiation or expansion media at different differentiation stages. The results demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and by HGF alone without G5-induced rES cell differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs; the early addition of HGF to EBs in the presence of G5 increased oligodendrocyte differentiation rate, but was not necessary, and the treatment during the first 2 days was enough to produce a similar effect; and HGF was required for terminal oligodendrocyte differentiation from NPs. Taken together, these results showed that HGF and G5 cooperatively promote rES cell differentiation into highly enriched oligodendrocyte precursors and mature oligodendrocytes.These observations set the method for obtaining highly enriched oligodendrocytes from ES cells in the nonhuman primate for clinical application and provide a platform to probe the molecular mechanisms that control oligodendrocyte differentiation.

Transcription factors Sox8 and Sox10 perform non-equivalent roles during oligodendrocyte development despite functional redundancy.

Development of myelin-forming oligodendrocytes in the central nervous system is dependent on at least two members of the Sox family of high-mobility-group-containing transcription factors. Sox9 is involved in oligodendrocyte specification, whereas Sox10 is required for terminal differentiation. We show that oligodendrocytes in the spinal cord additionally express the highly related Sox8. In Sox8-deficient mice, oligodendrocyte development proceeded normally until birth. However, terminal differentiation of oligodendrocytes was transiently delayed at early postnatal times. Sox8-deficient mice thus exhibited a similar, but less severe phenotype than did Sox10-deficient mice. Terminal oligodendrocyte differentiation was dramatically delayed in Sox8-deficient mice with only a single functional Sox10 allele hinting at redundancy between both Sox proteins. This redundancy was also evident from the fact that Sox8 bound to naturally occurring Sox10 response elements, was able to form DNA-dependent heterodimers with Sox10 and activated Sox10-specific oligodendrocytic target genes in a manner similar to Sox10. However, Sox8 expression levels were significantly lower than those for Sox10. Resulting differences in protein amounts might be a main reason for the weaker impact of Sox8 on oligodendrocyte development and for unidirectional compensation of the Sox8 loss by Sox10.

The Role of ErbB2 Signaling in the Onset of Terminal Differentiation of OligodendrocytesIn Vivo

The knock-out analyses of neuregulin and its receptors have indicated that they play essential roles in Schwann cell development. However, the role they play in oligodendrocyte development in vivo has remained unclear, because such knock-out animals die before CNS myelination begins. We examined the role of neuregulin signaling in the CNS by generating transgenic mice that express a dominant-negative mutant of the ErbB2 receptor among oligodendrocytes, using an MBP promoter. The transgenic mice exhibited widespread hypomyelination, resulting from a reduction in oligodendrocyte differentiation. The number of progenitors was conversely increased in the transgenic mice. We report that a reduction in oligodendrocyte differentiation is attributed in part to apoptosis of oligodendrocyte progenitors as they exit the cell cycle. A significant reduction in the number of p27+ oligodendrocyte precursors in the transgenic mice supports this conclusion. Taken together, these data suggest that for oligodendrocyte progenitors, ErbB2 signaling plays a role in governing a properly timed exit from the cell cycle during development into myelinating oligodendrocytes.

ErbB3 is dispensable for oligodendrocyte development in vitro and in vivo.

During development and in the adult, erbB2, erbB3, and erbB4 are expressed in many tissues and as heterodimers (B2/B3, B2/B4) serve as receptors for neuregulins. The general importance of neuregulin receptors for development is underlined by the observed embryonic (erbB2, erbB4) or perinatal (erbB3) lethality in mouse mutants. These mutants further revealed the fundamental role of the erbB2/erbB3 heterodimer for proper Schwann cell development, the ensheathing glia of the peripheral nervous system. However, only little is known about the functions of neuregulins and their receptors during postnatal development and in the adult. erbB2 and erbB3 during late embryogenesis and postnatally are expressed in different areas and cell types of the central nervous system, including oligodendrocytes, the ensheathing glia of the central nervous system. As terminal differentiation of oligodendrocytes peaks during postnatal development, it is not possible to use the neuregulin receptor mouse mutants to study terminal differentiation of oligodendrocytes in their absence in vivo. In order to investigate possible functions of the erbB3 gene in oligodendrocytes, we employed two different techniques. First, we directed the differentiation of erbB3-deficient embryonic stem cells into neural cell types to analyze the development of oligodendrocytes in the absence of erbB3 in vitro. Second, we grafted neural stem cells from spinal cords of erbB3 mutants into the retina of young mice to monitor oligodendrocyte differentiation and myelination in vivo. Results of both experimental approaches clearly show that erbB3 is not required for normal oligodendrocyte development and myelination.