Molecular mechanisms that control oligodendrocyte myelination during mammalian central nervous system (CNS) development are poorly understood. In this study, we identified Zfp488, an oligodendrocyte-specific zinc-finger transcription regulator, by screening for genes downregulated in the optic nerves of Olig1-null mice. The predicted primary structure of Zfp488 is evolutionarily conserved in vertebrates and invertebrates. In the developing CNS, Zfp488 is specifically expressed in oligodendrocytes but not their precursors. Its expression increases in parallel with that of major myelin genes Mbp and Plp1. Zfp488 is a nuclear protein that possesses transcriptional repression activity. In the developing chick neural tube, Zfp488 can promote oligodendrocyte precursor formation upon Notch activation. In addition, Zfp488 can interact and cooperate with the bHLH transcription factor Olig2 to promote precocious and ectopic oligodendrocyte differentiation. Furthermore, knockdown of Zfp488 via RNAi in an oligodendroglial cell line leads to the downregulation of myelin gene expression. Taken together, these data suggest that Zfp488 functions as an oligodendrocyte-specific transcription co-regulator important for oligodendrocyte maturation and that zinc-finger/bHLH cooperation can serve as a mechanism for oligodendroglial differentiation.

INTRODUCTION

Myelin-producing oligodendrocytes play a crucial role in supporting normal neuronal function of the mammalian CNS. Formation of myelinating oligodendrocytes from their precursors requires activation and coordination of a set of stage-specific transcriptional regulators that are important for the biosynthesis of myelin components (Gokhan et al., 2005; Kagawa et al.,2001; Wegner,2001). Although transcriptional regulation of oligodendrocyte precursor formation from neural progenitor cells is relatively well characterized in the developing CNS(Rowitch, 2004; Wegner, 2001), the molecular mechanisms governing oligodendrocyte maturation and myelinogenesis in postnatal CNS development are still poorly understood. The basic helix-loop-helix (bHLH) transcription factors Olig1/2 play a crucial role in oligodendrocyte differentiation and myelination as well as remyelination (Arnett et al.,2004; Lu et al.,2002; Xin et al.,2005; Yue et al.,2006; Zhou and Anderson,2002). Myelin formation in the postnatal CNS of Olig1-null (Olig1ΔKO) mice is severely compromised, despite the formation of oligodendrocyte precursor cells (OPCs)(Xin et al., 2005). At present, the downstream events mediated by Olig1 in oligodendrocyte myelination remain elusive.

To identify Olig1 downstream transcriptional regulators that may contribute to myelinating oligodendrocyte differentiation, we compared differential gene expression profiles between optic nerves from wild-type and Olig1-null mice (Xin et al.,2005). Optic nerves provide a naturally enriched source of myelinating oligodendrocytes as neural cell bodies in this tissue are exclusively glial, consisting mainly of those of myelinating oligodendrocytes and their precursors. By differential display analysis of genes downregulated in the optic nerves of Olig1 mutant mice(Liang and Pardee, 1992), we identified a previously uncharacterized nuclear zinc-finger transcriptional regulator Zfp488 that is specifically expressed in differentiating oligodendrocytes but not in their precursors. Zfp488 can interact and cooperate with Olig2 to induce ectopic and precocious oligodendrocyte differentiation in the developing chick neural tube. Furthermore RNAi-mediated Zfp488 knockdown leads to the downregulation of myelin genes in an oligodendroglial cell line. Thus, our studies suggest that Zfp488 is an oligodendrocyte-specific transcription modulator that cooperates with Olig2 to promote oligodendrocyte differentiation.

MATERIALS AND METHODS

Tissue collection and RNA in situ hybridization

Brains, spinal cords and optic nerves from wild type and Olig1mutants at various embryonic and postnatal stages were harvested from ketamine/xylazine anesthetized mice, fixed in 4% paraformaldehyde at 4°C overnight, infused with 20% sucrose in PBS overnight, embedded in OCT and cryosectioned at 16 μm. Digoxigenin-labeled riboprobes were used to perform RNA in situ hybridization, as described previously(Lu et al., 2002), and the probes used were: murine and chick Zfp488, Olig1, Olig2, Pdgfra,Plp1/Dm-20 (proteolipid protein) and Mbp (myelin basic protein). Detailed protocols are available upon request. Animal use and studies were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas.

RNA differential display and Northern blot:

Total RNA was harvested from the optic nerves from both wild-type and Olig1 knockout (Olig1ΔKO) mice from postnatal week 2. mRNA differential display (Liang and Pardee, 1992) was performed to identify and isolate differentially expressed genes according to the manufacturer's instructions(GeneHunter, TN). Northern blot was performed as previously described(Lu et al., 2000).

Quantitative real time polymerase chain reaction (QRT-PCR)

QRT-PCR was performed using the ABI Prism 7700 Sequence Detector System(Perkin-Elmer Applied Biosystems). RNA was extracted from cultured oligodendrocyte progenitor cells from neonatal rat forebrain and the CG4 oligodendroglial cell line (kindly provided by Dr Robin Miskimins) using Trizol (Invitrogen, Carlsbad, CA). cDNAs were generated using a first-strand cDNA synthesis kit (Amersham Bioscience, Piscataway, NJ). QRT-PCR was performed as previously described with Gapdh(glyceraldehyde-3-phosphatase dehydrogenase, TaqMan kit, Applied Biosystems)as an internal control (Xin et al.,2005). Primers used for expression analyses are as follows: Mbp, forward 5′-tcacagaagagaccctcaca-3′ and reverse 5′-gccgtagtgggtagttcttg-3′; Cnp, forward 5′-agctcaaggagaagaaccaat-3′ and reverse 5′-tgaagtgtcgaagctctttct-3′; Zfp488, forward,5′-ctcgagaaaatgacccttgt-3′ and reverse,5′-catgacagctctgtgaaggt-3′; Nkx2.2, forward 5′-catcgctacaagatgaaacg-3′ and reverse,5′-ctgtactgggcgttgtattg-3′.

Oligodendroglial cell cultures and siRNA transfection

Cortical oligodendrocyte precursors were isolated from neonatal rat at P2 as previously described (McCarthy and de Vellis, 1980; Yang et al.,2005). Oligodendrocyte precursor cells and CG4 oligodendroglial cells were maintained in serum-free medium containing N2 supplement(Invitrogen), 20 ng/ml FGF2 and 10 ng/ml PDGF-AA(Kondo and Raff, 2000; Raff et al., 1983). The medium was then switched to a medium containing 400 ng/ml Triiodothyronine T3 and 20 ng/ml ciliary neurotrophic factor (CNTF) to promote oligodendrocyte differentiation (Barres and Raff,1994; Gard and Pfeiffer,1989). SMARTpool siRNA (Dharmacon Catalog Number: M-088215-00 and accession number: XM_224697) is against four regions of rat Zfp488mRNA with 5′ starting positions of duplexes at 25, 250, 730 and 1285. Transfection of the SMARTpool siRNA into CG4 cells was performed by using lipofectamine 2000 (Invitrogen) per manufacturer's instructions.

Transient transfection, luciferase assay and immunohistochemistry

COS-7 and NIH 3T3 cells were seeded and grown in Dulbecco's modified Eagle medium with 10% fetal bovine serum one day before transfection. The cells reached 50-70% confluence on the day of transfection. Cells were transfected with LexA-Vp16, Zfp488 and its variants using FuGENE6 according to the manufacturer's protocol (Roche Applied Science, Indianapolis, IN) and assayed 48-hour post-transfection for luciferase activities by using Promega luciferase assay kit. In addition, the pRSV-beta-Galactosidase plasmid was included to control for variable transfection efficiencies between different experiments. The immunohistochemical staining procedure using anti-Myc antibody (Sigma, MO) for detection of Myc-Zfp488 and its derivatives for cellular localization was performed as described previously(Xin et al., 2005). Rabbit polyclonal antibody to Olig2 is kindly provided by Drs Chuck Stiles and John Alberta.

Co-immunoprecipitation and immunoblotting

COS7 cells were grown to about 60% confluence and then transiently transfected with 20 μg each of pCS2-MT:Zfp488 and pFLAG-CMV-6b:Olig2 by calcium phosphate precipitation. Whole cell lysates were prepared 48 hours after transfection by using 1× Passive Lysis Buffer (Promega, Madison, WI) supplemented with a protease inhibitor cocktail (1:200, Sigma, St Louis, MO). For immunoprecipitation, 600 μg of cell lysate proteins were incubated with 30 μg mouse anti-FLAG mAb in immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.5, 15 mM EGTA, 100 mM NaCl, 0.1% [w/v] Triton X-100, 1× protease inhibitor mixture, 1 mM DTT,1 mM PMSF) at 4°C for 4 hours. The antigen-antibody complex was collected by adding 40 μl (bed volume) of protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and incubating at 4°C for 2 hours. After three washes with IP buffer, the complex-bound resin was suspended in 1×SDS buffer, boiled and resolved on a 12.5% SDS-PAGE gel. After Western blotting, proteins carrying the Myc epitope tag were detected with mouse anti-Myc mAb (Clontech Laboratories, Palo Alto, CA) by using chemiluminescence with the ECL kit (Pierce, Rockford, IL.) according to the manufacturer's instructions. Monoclonal antibody to Nkx2.2 was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa under the auspices of the National Institute of Child Health and Human Development.

Chick embryo in ovo electroporation

Chicken eggs were incubated at ∼38°C. Approximately 1 μl (1.5μg/μl) of expression vectors carrying pCMV-GFP, Zfp488, Olig2,Nkx2.2, NICD or combinations thereof were injected into a chicken embryo neural tube at stage HH13-15 (E2.5) with the aid of Picospritzer III (Parker Hannifin, Cleveland, OH). The subsequent electroporation was performed by using a square wave electroporator (CUY21, BEX, Japan) with five pulses of electrical shock (25V, 50 mseconds for each pulse). Embryos were harvested 3 days after electroporation. At this stage (E5.5), none of oligodendrocyte markers is normally expressed in the neural tube. The green fluorescent segment of neural tube was dissected, fixed for 1 hour in 4%paraformaldehyde-PBS on ice and embedded in OCT for sectioning on a cryostat for in situ hybridization or immunohistochemistry. At least five embryos with expression of each transgene or their combination were analyzed and characterized.

RESULTS

Identification of an evolutionarily conserved zinc finger protein as a gene downstream of Olig1

To identify transcriptional regulators of myelinating oligodendrocyte formation representing potential downstream targets of Olig1, we screened for genes downregulated in the optic nerves of 14-day old Olig1 null mice using differential display. In addition to previously identified genes known to encode myelin components or their regulators, a cohort of novel transcripts downregulated or undetectable in the Olig1 mutant were identified (Fig. 1A, data not shown). Among them, a cDNA encoding a zinc-finger motif-containing protein was identified as Zfp488 (zinc-finger protein for oligodendrocyte differentiation). The predicted amino acid sequence of Zfp488 (GenBank Accession Number XM_356719) is 52% identical and 64%homologous to that of a putative gene entered as zinc-finger protein 488 in the human genome database (GenBank Accession Number BC 051323). Northern blot analysis (Fig. 1B) indicates that Zfp488 is a ∼3.5 kb transcript absent from the brain of the Olig1 mutant, consistent with the differential display result.

The Zfp488 mRNA encodes a 337 amino acid peptide with two typical C2H2 zinc finger motifs at its C terminus. A bipartite nuclear localization signal (NLS) is located between them (Fig. 1C). To determine whether Zfp488 has counterparts in other species, a homology search revealed that murine Zfp488 appears to have an ortholog but no obvious paralogs in species, including human, rat,chicken and Drosophila (Fig. 1D), suggesting a conserved non-redundant function for Zfp488 during evolution. The structural feature with two zinc-finger motifs flanking an NLS is highly conserved across all species(Fig. 1D).

Zfp488 is specifically expressed in differentiated oligodendrocytes in the developing CNS

To determine the cell type(s) that express Zfp488 in the CNS, we compared the expression pattern by performing in situ hybridization for Zfp488 and stage-specific oligodendroglial lineage genes in the developing murine spinal cord, where oligodendrocyte development is relatively well characterized. Although the oligodendrocyte precursor marker Pdgfra is present in the ventral spinal cord at embryonic day 12.5(E12.5) (Fig. 2B), expression of neither Zfp488 nor the oligodendrocyte differentiation markers Mbp and Plp1/DM20 is detected at this stage(Fig. 2A,C; data not shown). Zfp488 is initially expressed as foci in the ventral domain of the spinal cord at E14.5 (Fig. 2D),which coincides with the expression of Mbp and Plp1/DM20(Fig. 2E,F). At the late embryonic stage E18.5, Zfp488 expression appears to extend to the white matter of the spinal cord, concurrent with Mbp and Plp1/DM20 expression in this region(Fig. 2G, compare with 2H,I). By contrast, Zfp488 expression is not observed in peripheral myelinating Schwann cells in the dorsal root ganglion (drg)(Fig. 2D, compare with 2E, blue arrows).

In the postnatal developing CNS, Zfp488 expression is highly enriched in the white matter tracts of the spinal cord, the forebrain and the cerebellum (Fig. 3A-C). In addition, a population of Zfp488+ cells is also detected in the gray matter of the spinal cord and the forebrain(Fig. 3A,B; black arrows). In the optic nerve and the corpus callosum, Zfp488-expressing cells can form a linear array (Fig. 3D,E), a characteristic feature of interfascicular myelinating oligodendrocytes.

To further determine the identity of Zfp488 expressing cells, we performed double in situ hybridization of Zfp488 with markers for differentiated oligodendrocytes or their precursors in the brain at P14. The majority of Zfp488+ cells co-express Plp1 in the corpus callosum (Fig. 3E). Similar co-expression of Zfp488 and Mbp was also observed (data not shown). By contrast, Zfp488 expression is essentially absent in oligodendrocyte precursors marked by Pdgfra expression(Fig. 3F), suggesting Zfp488 expression is largely confined to differentiated oligodendrocytes.

To determine whether Zfp488 is genetically downstream of Olig1 throughout the CNS, we examined its expression in Olig1 mutant mice. In this mutant, Zfp488 expression is undetectable in the spinal cord and brain regions of both embryonic(Fig. 4A,C,E) and postnatal(Fig. 4B,D,F) stages. As Olig1 mutant mice fail to form mature oligodendrocytes but do form OPCs, the loss of Zfp488 expression in Olig1-null mice is consistent with the notion that Zfp488 is mainly expressed in differentiated or mature oligodendrocytes.

To test whether Olig1 can regulate Zfp488 transcription,we co-transfected Olig1 and a luciferase reporter driven by 3.2 kb Zfp488 upstream regulatory region, which contains 22 consensus E-boxes potentially recognized by bHLH transcription factors. Olig1appears to transactivate the reporter driven by this candidate regulatory region approximately fourfold (Fig. 4G). Although Olig2 activates the putative promoter to a lesser extent, the difference is not statistically significant(Fig. 4G). Thus, our data suggest that Zfp488 expression can be directly or indirectly regulated by Olig1.

Zfp488 exhibits nuclear localization and has transcriptional repression activity

In silico analysis revealed that Zfp488 contains a possible NLS sequence: a bipartite NLS between its two zinc-finger motifs(Fig. 1C). To examine the subcellular localization of Zfp488, Myc-tagged Zfp488 was cloned into a mammalian expression vector and transfected into NIH3T3 and COS7 cell lines. Myc-tagged Zfp488 expression was consistently observed in the nucleus defined by co-localization of DAPI and Myc immunoreactivity in these cell lines(Fig. 5A,B). To delineate the region responsible for nuclear localization, we performed a domain mapping experiment, wherein a series of truncation or deletion mutants were generated. The subcellular localization of these mutants was examined after transfection into NIH3T3 cells. All truncated Zfp488 mutants lacking the bipartite NLS motif were exclusively cytoplasmic (Fig. 5D,E). By contrast, the mutant carrying only two zinc finger motifs and the NLS can be detected in the nucleus(Fig. 5F), suggesting that this NLS is likely responsible for nuclear localization of Zfp488.

To determine the transcriptional activity of Zfp488, we used an in vitro Vp16 transcription activation reporter system carrying adjacent LexA and GAL4-binding sites (Fig. 5G)(Lu et al., 1999). Although the LexA-VP16 fusion protein activated reporter expression markedly, the GAL4-Zfp488 fusion protein alone did not alter the reporter activity. However,when GAL4-Zfp488 was co-transfected with LexA-VP16, the binding of GAL4-Zfp488 adjacent to the LexA-VP16 activator led to an approximately eightfold reduction of VP16 activation(Fig. 5H), suggesting that Zfp488 possesses repressive transcriptional activity.

To further define the region responsible for this repressive activity, a series of truncation or deletion mutants were generated as GAL4 fusions. The repressive effects of these mutant forms of Zfp488 were examined and compared(Fig. 5H). Deletion of zinc-finger motifs (up to residue 184 in the C-terminal) did not abolish the repressive effects. By contrast, further deletion of amino acids 69-184 eliminates the ability of Zfp488 to inhibit VP16-mediated activation. In addition, expression of the zinc-finger domain alone did not affect Vp16 trans-activation (Fig. 5H). Thus, our results suggest that a potential repression domain resides in the segment spanning amino acids 69-184 of the Zfp488 protein outside the zinc-finger domains.

Zfp488 promotes oligodendrocyte precursor formation in the presence of Notch signaling activation

To examine whether ectopic expression of Zfp488 in vivo could promote oligodendrocyte generation, we carried out a gain-of-function study in the developing chick neural tube (Sun et al., 2001; Zhou et al.,2001). A Zfp488 expression vector was electroporated into the neural tube of E2.5 chick embryos. The embryos were then harvested 3 days later at E5.5, when the differentiation of endogenous oligodendrocytes has not yet occurred (Ono et al.,1995). Misexpression of Zfp488 alone did not induce ectopic oligodendrocyte differentiation in the neural tube(Fig. 6A,B). One possibility is due to the endogenous and prevailing proneural activity at this stage(Zhou et al., 2001). To repress the neurogenic activity, we misexpressed Zfp488 together with NotchICD (NICD), a constitutively active form of Notch. NICD is known to repress a number of proneural genes while instructing and/or permitting gliogenesis in vertebrate systems(de la Pompa et al., 1997; Ma et al., 1996; Morrison et al., 2000; Park and Appel, 2003). As a control, overexpression of NICD itself did not induce oligodendrocyte precursor formation (Fig. 6C,D). However, misexpression of Zfp488 with NICD in the chick neural tube was able to promote ectopic expression of committed oligodendrocyte precursor markers Pdgfra and Sox10 on the electroporated side of the neural tube(Fig. 6E-G). The ectopic expression of OPC markers was mainly detected in the ventricular zone of both the dorsal and ventral neural tube (Fig. 6E-G, arrowheads). No mature oligodendrocyte markers such as Mbp, however, were detected (Fig. 6H). These data suggest that Zfp488 is able to promote precocious oligodendrocyte precursor formation upon Notch signaling activation.

Co-misexpression of Zfp488 and Olig2 induces ectopic and precocious oligodendrocyte differentiation

The absence of ectopic Mbp expression in the Zfp488/NICDco-electroporation assay as described above might reflect the inhibitory effect of Notch signaling on oligodendrocyte maturation(Wang et al., 1998). Alternatively, Zfp488 might need to cooperate with additional transcriptional regulators to promote terminal differentiation of OPCs. As Olig2 and Nkx2.2 are involved in promoting oligodendrocyte differentiation (Qi et al.,2001; Zhou et al.,2001), we therefore examined whether misexpression of Zfp488 with these oligodendroglial regulators could promote ectopic oligodendrocyte differentiation.

Expression vectors carrying Zfp488, Olig2 or both were electroporated into E2.5 chick embryos harvested and analyzed 3 days later at E5.5. Misexpression of Olig2 alone did not lead to ectopic oligodendrocyte formation in the dorsal spinal cord(Fig. 7A,B) despite the appearance of a small population of cells expressing Sox10 in the ventral domain. By contrast, co-electroporation of Zfp488 and Olig2resulted in robust induction of ectopic and precocious expression of Sox10 and Pdgfra (Fig. 7C-E) on the electroporated side of the chick neural tube. Significantly, co-expression of Zfp488 and Olig2 promotes ectopic expression of Mbp in this region(Fig. 7F). Many of these ectopic oligodendrocytes appeared to be in the dorsal gray matter away from the ventricular zone, suggesting that they differentiated. By contrast,co-misexpression of Zfp488 and Nkx2.2 did not induce ectopic oligodendrocyte formation (Fig. 7G,H).

To avoid ectopic oligodendrocyte formation owing to interaction of Olig2 with endogenous Nkx2.2 in the ventral spinal cord,dorsal expression of Olig2 and Zfp488 was achieved by dorsally oriented in ovo electroporation(Fig. 7I). Under this circumstance, ectopic Sox10 was detected ectopically in the dorsal region of the spinal cord (Fig. 7J, red arrows), although there was no ectopic expansion of this oligodendroglial marker into the ventral domain of the spinal cord(Fig. 7J, black arrow). Thus,Zfp488 appears to cooperate with Olig2 to promote ectopic and precocious oligodendrocyte differentiation.

Zfp488 is co-expressed and physically interacts with Olig2

To examine whether Zfp488 and Olig2 are colocalized in the same cells, we performed double in situ hybridization and immunohistochemistry on the chick spinal cord at the stage E18. Expression of endogenous chick Zfp488 is mainly confined to the white matter of the spinal cord(Fig. 7K). Essentially all Zfp488-expressing cells are Olig2 positive(Fig. 7L, arrows), although not all Olig2+ cells express Zfp488(Fig. 7L). Thus, co-expression of Zfp488 with Olig2 is consistent with the cooperation of Zfp488 and Olig2 in promoting oligodendrocyte differentiation.

As Olig2 and Zfp488 are co-expressed in oligodendrocytes and cooperate to promote oligodendrocyte differentiation, we then examined whether these two factors can associate with each other. Zfp488,Olig2 or both expression vectors were transfected into COS7 cells. Co-immunoprecipitation (IP) was performed from the cell lysates 48 hours after transfection. Olig2 was detected in the immunoprecipitated complex containing Zfp488 (Fig. 7M), suggesting that Zfp488 can either directly interact with Olig2 or that they are components of a protein complex. By contrast, Zfp488 did not appear to interact with Nkx2.2 in this assay (Fig. 7N).

Zfp488 expression is upregulated as oligodendroglial cells become differentiated

Our data suggest that Zfp488 is involved in the late stage of oligodendrocyte differentiation and maturation by interacting with Olig2,while a previous study indicates that Nkx2.2 interacts with Olig2 at an early stage for oligodendrocyte fate determination(Zhou et al., 2001). To define the relation between Zfp488, Nkx2.2 and myelin gene expression, we examined expression of these genes during the progression of oligodendrocyte differentiation in primary culture. OPCs were isolated from the neonatal rat brain and cultured in growth medium in the presence of PDGFAA and bFGF(Kondo and Raff, 2000; Raff et al., 1983). Differentiation was induced by switching to oligodendrocyte differentiation medium containing T3 and CNTF (Barres and Raff, 1994; Gard and Pfeiffer,1989). Total RNA from these cultures was isolated at different time points before and after the induction of oligodendrocyte differentiation and subjected to quantitative real-time PCR analysis. Upon OPC differentiation, Zfp488 expression was upregulated rapidly in conjunction with increased expression of oligodendrocyte differentiation markers Mbp and Cnp (2′,3′-cyclic nucleotide 3′-phosphohydrolase) (Lappe-Siefke et al., 2003) (Fig. 8A). This is in contrast to the downregulation of Nkx2.2during oligodendrocyte differentiation(Fig. 8A)(Qi et al., 2001; Wei et al., 2005). Thus, the level of Zfp488 expression accumulates in parallel with that of myelin genes, and is inversely correlated with that of Nkx2.2 during oligodendrocyte maturation.

RNAi-mediated Zfp488 knockdown results in a decrease in myelin gene expression

To determine the effects of Zfp488 knockdown on myelin gene expression, we used RNAi targeting Zfp488 in CG4 cells, a cell line with properties of rat immature oligodendrocytes(Espinosa de los Monteros et al.,1997; Tontsch et al.,1994; Wei et al.,2005). Zfp488 expression was rapidly upregulated when CG4 cells were in differentiation medium. This is correlated with the expression of Mbp and Cnp but negatively related to Nkx2.2expression (Fig. 8B). This observation recapitulates the pattern of Zfp488 expression seen during primary oligodendrocyte differentiation, suggesting that CG4 cells exhibit normal overall oligodendroglial gene regulation. To knock down Zfp488 mRNA, short interfering RNA (siRNA) duplexes were designed against rat Zfp488. Transfection of CG4 cells with Zfp488siRNAs led to a reduction of ∼60% of Zfp488 expression examined by quantitative real time-PCR analysis(Fig. 8C). The down-regulation of Zfp488 caused a decrease in the expression level of endogenous myelin genes Mbp and Cnp in CG4 cells, but not the control housekeeping gene Gapdh. By contrast, the control siRNA against an unrelated gene GFP did not reduce Zfp488 expression and myelin gene expression (Fig. 8C, data not shown). These data therefore suggest that specific knockdown of Zfp488 in the oligodendroglial cell line leads to the downregulation of myelin gene expression, consistent with a role for Zfp488 in promoting oligodendrocyte maturation.

DISCUSSION

Oligodendrocyte myelinogenesis is characterized by successive stages of lineage progression from precursors to immature oligodendrocytes and finally to mature myelinating oligodendrocytes(Pfeiffer et al., 1993). Although a series of transcriptional regulators has been found in oligodendrocyte precursors (Rowitch,2004), few have been identified that are specifically expressed in mature oligodendrocytes in the CNS. In the peripheral nervous system, a zinc-finger transcription factor Krox20, which is restricted to mature myelinating Schwann cells, plays an essential role in Schwann cell myelination(Topilko et al., 1997; Topilko et al., 1994). Thus,identification of transcriptional regulators that are spatially and temporally limited to myelinating oligodendrocytes might contribute to unraveling the molecular mechanisms that control the oligodendrocyte myelination process.

In a screen for mRNAs downregulated in non-myelinating optic nerves of Olig1-null mice, we identified an as yet uncharacterized zinc-finger transcription regulator Zfp488. It is specifically expressed in oligodendrocytes at later stages of differentiation but not in myelinating Schwann cells in the peripheral nervous system. Ectopic expression of Zfp488 together with Notch activation or in collaboration with Olig2 induces precocious oligodendrocyte differentiation in the chick neural tube. In addition, RNAi-mediated Zfp488 knockdown leads to myelin gene downregulation in an oligodendroglial cell line. Thus, our gain- and loss-of-function data suggest that Zfp488 probably plays an important role in the differentiation and maturation process during oligodendrocyte development.

The oligodendrocyte-specific zinc finger protein Zfp488, a downstream effector of Olig1, cooperates with Olig2 to promote oligodendrocyte differentiation

Expression of Zfp488 persists in oligodendrocytes but not their precursors throughout the CNS, indicating that Zfp488 is specific for differentiated oligodendrocytes. The observations that Zfp488expression is undetectable in the brain and spinal cord of Olig1mutant mice, and that Olig1 transactivates a candidate Zfp488 regulatory sequence, suggest that Zfp488 is a direct or indirect downstream target gene of the transcription regulator Olig1.

The cooperation between oligodendrocyte-specific Zfp488 and Olig2 induces ectopic and precocious oligodendrocyte maturation, suggesting that Zfp488 functions as a transcriptional co-regulator for oligodendrocyte differentiation and maturation. Several lines of evidence indicate that interaction of zinc-finger proteins with proneural bHLH proteins is a common theme for inducing neuronal cell fate specification(Acar et al., 2006; Bellefroid et al., 1996; Nakakura et al., 2001). Overexpression of X-Myt1, a Xenopus zinc-finger protein, promotes ectopic neuronal differentiation only in combination with proneural bHLH transcription factors (Bellefroid et al.,1996). Recently, Acar et al. showed that a Drosophilazinc-finger transcription factor, Senseless, is recruited by bHLH proneural proteins to function as a co-activator in promoting the development of sensory organ precursors (Acar et al.,2006). In keeping with these observations, the physical and functional interaction of the zinc finger protein Zfp488 and the bHLH factor Olig2 in promoting oligodendroglial maturation may reflect a similar mechanism for zinc finger/bHLH cooperation in regulating glial differentiation during development.

Zfp488 exerts repressive effects on transcription and promotes oligodendrocyte precursor formation upon Notch activation

Zfp488 is a nuclear protein in the different cell lines tested. Like many oligodendrocyte transcriptional regulators, including Olig2, Nkx2.2 and Nkx6.2(Awatramani et al., 2000; Wei et al., 2005; Zhou et al., 2001), Zfp488 also functions apparently as a transcriptional repressor when assayed by an in vitro transcription reporter system. Its transcriptional repression domain appears localized outside the zinc-finger motifs, suggesting that the repressive regulatory domain in Zfp488 is separate from a potential DNA recognition domain mediated by zinc-finger motifs. It is possible that the repression activity common among oligodendrocyte transcriptional regulators may be required for directing oligodendrocyte differentiation while repressing neurogenic activity during development.

Although overexpression of Zfp488 itself cannot promote oligodendrocyte differentiation, Zfp488 is able to promote precocious oligodendrocyte precursor formation in the chick neural tube under the condition of constitutive Notch activation. One potential mechanism is that activation of Notch signaling may provide an environment to facilitate Zfp488 in promoting oligodendroglial specification either by repressing neurogenic activity of proneural proteins, as occurs when Notch activation facilitates promotion of OPC formation by Olig2 (Zhou et al.,2001) or by destabilizing proneural bHLH proteins(Sriuranpong et al., 2002). These data suggest a more general underlying mechanism where Notch activation provides a permissive environment for transcription regulators to induce oligodendrocyte precursor specification.

Dynamic coupling of stage-specific transcription regulators may control successive waves of oligodendrocyte maturation during CNS development

Olig2 is essential for oligodendrocyte lineage development. Its expression is detected in OPCs during early CNS development and persists in mature myelinating oligodendrocytes in adulthood. At present it is not fully understood how the transition from oligodendrocyte precursors to mature myelinating oligodendrocytes is regulated by Olig2 at different developmental stages (Pfeiffer et al., 1993; Rowitch, 2004). A recent study suggests that an interplay between transcriptional activators and inhibitory factors may orchestrate myelin gene expression(Gokhan et al., 2005; Kagawa et al., 2001; Wegner, 2001). In the developing neural tube, the transient overlap of Olig2 and Nkx2.2 expression domains in the ventricular zone specifies oligodendrocyte precursors,suggesting that Olig2 cooperates with Nkx2.2 for the early specification of OPCs (Zhou et al., 2001). However, the expression of Nkx2.2 is downregulated in oligodendroglial cells undergoing terminal differentiation into myelinating oligodendrocytes(Watanabe et al., 2004; Wei et al., 2005)(Fig. 8). There is also evidence that Nkx2.2 represses Mbp expression in oligodendrocytes in vitro (Wei et al., 2005). Thus, the effects of Nkx2.2 may depend on its temporal context, i.e. Nkx2.2 may cooperate with Olig2 for oligodendrocyte differentiation at an early developmental stage while repressing oligodendrocyte maturation at later stages.

Zfp488 is upregulated as immature oligodendroglial cells become differentiated. Increased Zfp488 may subsequently interact with Olig2 to promote oligodendrocyte terminal differentiation. The functional interaction between Zfp488 and Olig2 correlates well with their co-expression in oligodendrocytes. Expression of Zfp488 specifically in differentiated and mature oligodendrocytes, coupled with downregulation of Nkx2.2 during oligodendrocyte maturation, suggests that Zfp488 may potentially cooperate with Olig2 at a later stage of differentiation to promote oligodendrocyte maturation and myelination. Thus, our study supports the notion that dynamic coupling of stage-specific oligodendroglial regulators may control successive waves of oligodendrocyte maturation during CNS development.

Acknowledgements

This study was funded by grants from the National Multiple Sclerosis Society, Wadsworth Foundation, March of Dimes Birth Defect Foundation, and National Institutes of Health (R01NS050389 to QRL). We thank Drs Steve Kernie,Jenny Hsieh, Jane Johnson and Robin Miskimins for comments and Kendy Xian for technical assistance. QRL is a Harry Weaver Neuroscience Scholar and a Basil O'Connor Scholar.

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