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First published online August 14, 2006
doi: 10.1242/10.1242/dev.02522
Center for Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
* Author for correspondence (e-mail: qrichard.lu{at}utsouthwestern.edu)
Accepted 7 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Oligodendrocyte myelination, Zinc finger protein, bHLH transcription factors, Olig1, Olig2, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 (Olig1
KO) 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 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 (Olig1KO) 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 Zfp488
mRNA 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 1x 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, 1x 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 1x
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 (1.5
µg/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.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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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. Olig1 appears 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.
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). 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/NICD
co-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 Olig2 resulted 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).
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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 Nkx2.2
during 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 Nkx2.2
expression (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 Zfp488
siRNAs 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 |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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 Zfp488
expression is undetectable in the brain and spinal cord of Olig1
mutant 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.
|
;
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 Drosophila
zinc-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.
|
;
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.
|
;
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.
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ACKNOWLEDGMENTS |
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