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First published online 11 April 2007
doi: 10.1242/dev.02847
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1 Department of 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.
2 Department of Psychiatry, University of Texas Southwestern Medical Center,
Dallas, TX 75390, USA.
3 Department of Pediatrics, University of Texas Southwestern Medical Center,
Dallas, TX 75390, USA.
4 Department of Molecular Biology, University of Texas Southwestern Medical
Center, Dallas, TX 75390, USA.
* Author for correspondence (e-mail: qrichard.lu{at}utsouthwestern.edu)
Accepted 7 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Astrocyte differentiation and heterogeneity, Oligodendrocyte lineage, bHLH transcription factors, Olig2, Cortex, Spatiotemporally specific knockout, mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Shu and Richards, 2001;
Sofroniew, 2005;
Ullian et al., 2001). Cortical
astroglial cells are derived from neural progenitors, including radial glia
present in the ventricular zone (VZ) and subventricular zone (SVZ) of the
developing brain at embryonic and early postnatal stages
(Goldman, 2004;
Miller and Reynolds, 2004)
that migrate into the cortex, where they differentiate and become mature
astrocytes at adulthood (Sauvageot and
Stiles, 2002; Temple,
2001). Astrocytes in the cortex exhibit heterogeneous morphologies
and are subdivided into at least two subpopulations - white and gray matter
astrocytes - based on their location. White matter astrocytes harbor abundant
glial filaments in their processes, whereas gray matter astrocytes exhibit
many branched processes and few glial filaments. These spatially distinct
astrocyte subclasses are commonly defined by expression of glial fibrillary
acidic protein (GFAP) (Kimelberg,
2004; Reichenbach and Wolburg,
2005; Walz, 2000).
Although many signaling pathways involved in astrocyte differentiation in
vitro have been identified (Mehler,
2002; Sauvageot and Stiles,
2002; Sun et al.,
2003), at present it is unclear whether the formation of spatially
and morphologically heterogeneous astrocytes is subject to identical or
distinct transcriptional regulatory mechanisms.
The basic helix-loop-helix (bHLH) transcription factor Olig2 is essential
for the formation of motoneurons and oligodendrocytes in the developing spinal
cord (Lu et al., 2002;
Takebayashi et al., 2002;
Zhou and Anderson, 2002);
however, its functions in astrocyte development remain elusive, particularly
in the developing brain. Olig2 expression can be detected in cortical neural
progenitors including radial glial cells at embryonic stages and in
GFAP+ progenitor/stem cells in the postnatal SVZ
(Furusho et al., 2006;
Malatesta et al., 2003;
Setoguchi and Kondo, 2004).
Recent studies with a dominant-negative form of Olig2 indicate that Olig2
directs cortical astrocyte differentiation from neural progenitors in the SVZ
of the neonatal brain (Marshall et al.,
2005), whereas in vitro evidence suggests that Olig2 inhibits
astrocyte differentiation (Fukuda et al.,
2004; Gabay et al.,
2003). In vivo characterization of the function of Olig2 in
cortical astroglial differentiation, which occurs largely at postnatal stages
(Sauvageot and Stiles, 2002),
has been hampered by the early embryonic lethality of Olig2-null mice
(Lu et al., 2002;
Takebayashi et al., 2002;
Zhou and Anderson, 2002). At
present, it is not clear how Olig2 regulates the formation of distinct
astrocyte subpopulations in the developing brain.
In this study, we show that Olig2 is transiently expressed in immature developing astrocytes neonatally and its level is downregulated progressively in mature astrocytes at late postnatal stages. We utilize a spatiotemporally specific conditional ablation approach to examine Olig2 function in astrocyte development in the postnatal CNS. We uncover a crucial role of Olig2 in white matter astrocyte differentiation in the brain and the spinal cord. By contrast, in the gray matter, Olig2 ablation leads to abnormal GFAP upregulation in a population of astrocytes located in superficial cortical layers. Cell type-specific ablation and fate-mapping analyses indicate that abnormal development of cortical astrocytes is at least in part attributable to the loss of Olig2 function. Thus, our studies using conditional in vivo mutagenesis indicate an important dual role of Olig2 in regulating gray and white matter astrocyte development and reveal distinct transcriptional requirements for the formation of diverse astrocyte subpopulations during CNS development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
induce Olig2 ablation, 0.3 mg TM per 4 g body weight was injected
intraperitoneally into neonatal pups and their foster mother as described
previously (Hayashi and McMahon,
2002). All protocols involving the use of animals were approved by
the Institutional Animal Care and Research Advisory Committee (IACRAC) at the
UT Southwestern Medical Center at Dallas.
RNA in situ hybridization and immunohistochemistry
RNA in situ hybridization and probes, as well as immunostaining methods
with tissue sections from mouse brains, were as described previously
(Lu et al., 2002). Double
immunostaining was performed by simultaneous incubation with the relevant
antibodies. The following antibodies were used: Olig2 (gift from Chuck Stiles,
Harvard Medical School, Boston, MA); GFAP (Dako), NG2 and NeuN (Chemicon),
BrdU, GFAP, S100ß, NF200, Tau-1, Gap43 (Sigma), Ki67 (1:500, Swant) and
rat PDGF
R (1:500, BD Bioscience). For
5'-bromo-2'-deoxyuridine (BrdU) immunostaining, perinatal control
and mutant littermates were injected with 100 mg BrdU (Sigma, St Louis, MO)
per kg body weight 4 hours prior to sacrifice. Goat anti-mouse, rat and rabbit
secondary antibodies conjugated to Cy2 and Cy3 (Jackson ImmunoResearch) were
used for double-labeling experiments. Microscopy was performed on a Zeiss LSM
510 laser scanning confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Fig.
1C-E). Whereas GFAP expression became undetectable in the cortex
at perinatal stages such as P14 (Figs
2 and
3), a low level of Olig2
expression was detectable in GS+ cortical astrocytes
(Fig. 1H,I, arrowheads), as
compared with intense Olig2 expression in early-developing astrocytes
(Fig. 1F,G). At this stage,
Olig2+ GS- cells with a high level of Olig2 expression
were only identified as oligodendrocyte lineage cells
(Fig. 1H,I, arrows; data not
shown). Olig2 expression was completely absent from GS+ cortical
astrocytes of young adult mice by P21 (Fig.
1J,K). Similarly, in the corpus callosum - a developing cerebral
white matter tract - of wild-type mice, Olig2 expression was detected in a
population of developing GFAP+ astrocytes at P5
(Fig. 1L-O), but was absent in
mature astrocytes by adulthood (data not shown). Thus, Olig2 appears to be
transiently expressed in immature developing GFAP+ astrocytes at
early postnatal stages, whereas it undergoes downregulation in mature
astrocytes at late postnatal stages and adulthood.
Abnormal astrocyte development in the Olig2-ablated cortex
To assess the function of Olig2 in cortical astrocyte formation,
we used a hGFAP-Cre deleter line (Zhuo et
al., 2001) to ablate Olig2 in the developing cortex by
generating Olig2Cko;hGFAPCre (CkoG)
mice. The hGFAP promoter drives Cre recombinase expression in the majority, if
not all, embryonic multipotent cortical progenitors, which give rise to neural
cell types including neurons, oligodendrocytes and astrocytes in the cortex
(Malatesta et al., 2003;
Zhuo et al., 2001). In
addition, the Cre activity persists in developing astrocytes and SVZ
precursors at postnatal stages (Malatesta
et al., 2003; Zhu et al.,
2005).
In control mice, Gfap was readily detected in the cerebral white
matter, the hippocampus and at the subpial surface. However, expression of
Gfap, as assessed by in situ hybridization, was essentially
undetectable in the cortical gray matter from postnatal stage P7 to adulthood
(Fig. 2A,C,G), although a
population of GFAP+ cells was detected in the cortex at early
neonatal stages (Fig. 1). These
observations are consistent with previous observations that most gray matter
astrocytes contain little, if any, GFAP
(Connor and Berkowitz,
1985).
By contrast, in the Olig2 conditional mutant
(CkoG) mice, intense Gfap expression was
initially observed in cells scattered throughout the cortex at neonatal stage
P7 (Fig. 2B), and increased
dramatically from postnatal week 2 (Fig.
2D). High Gfap expression persists throughout adulthood
(Fig. 2H). This increase in
expression was not uniform in the Olig2-ablated cortex. The highest
level of Gfap expression was seen in superficial cortical layers
II-IV and the stratum lacunosum-moleculare of the hippocampus
(Fig. 2D,F), a region
anatomically akin to the superficial layers of the cortex
(Forster et al., 2006), whereas
Gfap expression in the deep layers of the cortex (layers V-VI) was
essentially undetectable (Fig.
2D,H). Strikingly, in contrast to Gfap upregulation in
the gray matter, Gfap expression in the white matter was
significantly downregulated in Olig2 mutants at perinatal and adult
stages (Fig. 2F,H compared with
E,G, arrowheads). The lower level of in situ signal could be due
to fewer astrocytes or reduced GFAP expression in each cell. Nonetheless,
these observations suggest that the effect of Olig2 ablation on
Gfap expression is region-specific within the cortex.
Astrocyte formation deficit in the white matter of the Olig2-ablated cortex
In the cerebral white matter of Olig2-ablated mice, we observed
marked downregulation not only of Gfap mRNA but also at the protein
level, as compared with the control (Fig.
3A-D, arrowhead). When Olig2-ablated cortices were
examined by Hematoxylin and Eosin staining for cell morphology, there was a
substantial reduction of cell number in the corpus callosum
(Fig. 3F compared with E). To
determine whether the formation of astrocytes is compromised in this region,
we examined expression of another astrocyte marker, S100ß. In the white
matter of Olig2 mutants, the number of S100ß+ cells
was severely reduced, in contrast to the high density of these cells in the
control (Fig. 3G,H,O). Since
S100ß could reflect a population of oligodendrocyte lineage cells, we
examined expression of an additional astrocyte marker, GS. The number of
GS-expressing cells in the corpus callosum of Olig2 mutants
(Fig. 3M,N) was markedly
reduced to approximately 21% of wild-type and control hGFAP-Cre
(CtrlG) mice (Fig.
3I-L,O). Expression of the astrocyte markers GS and GFAP in the
white matter of wild-type and hGFAP-Cre-expressing control mice
(CtrlG) (Fig. 3I,J
compared with K,L) was comparable
(Fig. 3O), suggesting that
expression of hGFAP-Cre does not exert detectable toxic effects on the
formation of white matter astrocytes. A disproportionate reduction in
S100ß+ cells as compared with GS+ cells
(Fig. 3O) is likely to be due
to a population of S100ß+ cells marking early-differentiated
oligodendrocytes (Deloulme et al.,
2004; Rickmann and Wolff,
1995) that are lost in the Olig2 mutant
(Yue et al., 2006). Thus, the
reduction in cell number and the expression of different astrocyte markers
suggest that Olig2 is required for formation and differentiation of an
astrocyte subpopulation in the cerebral white matter.
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; Yue et al.,
2006), but also persists postnatally in GFAP+
developing astrocytes in the white matter and in GFAP+ progenitors
in the SVZ (Fig. 4A-D). It has
been suggested that astrocyte subtypes in the optic nerve have distinct
temporal maturation patterns (Miller and
Raff, 1984). To determine the critical stage at which
Olig2 function is needed for cerebral white matter astrocyte
formation, we ablated Olig2 at early postnatal stages by breeding
Olig2 conditional mutant mice with an inducible Cre line
(CAGGS-CreERTM) (Hayashi and
McMahon, 2002), in which tamoxifen (TM)-inducible Cre activity is
controlled by a ubiquitous cytomegalovirus promoter.
The control and compound Olig2Cko;CreERTM animals were
administrated TM at neonatal stages for 4 days, from P2-P5, and their brains
harvested at P13 for in situ hybridization analysis. Olig2 expression
in the cortex was essentially eliminated by TM-induced Cre activity
(Fig. 4F). Since Olig2 is
required for adequate postnatal myelination
(Yue et al., 2006), expression
of the myelin gene Mbp and of the mature oligodendrocyte marker CC1
were accordingly significantly downregulated in the gray and white matter of
the Olig2-ablated cortex, as compared with the TM-injected control
(Fig. 4G,H,M). Residual
Mbp+ cells in the white matter are likely to result from
the continued maturation of developing oligodendrocytes formed prior to
TM-induced Olig2 ablation in these cells. In the inducible
Olig2 mutant, Gfap expression was severely downregulated in
the cerebral white matter as compared with the otherwise robust expression in
this tract, whereas Gfap expression persisted in the SVZ and subpial
surface at a level comparable to the control
(Fig. 4J,L compared with
I,K).
Downregulated expression of other astrocyte markers such as GS was also observed in the cerebral white matter of the TM-treated Olig2 mutant mice (Fig. 4M). By contrast, Gfap expression was not perturbed in the cortical gray matter (Fig. 4J) and there was no significant difference in terms of the number of GS+ astrocytes in the cortical areas when Olig2 was ablated postnatally (Fig. 4M). These results suggest that TM-induced Olig2 deletion at neonatal stages results in a severe deficit in the formation of white matter astrocytes. Thus, the results from inducible Olig2 ablation suggest that Olig2 plays an important role in postnatal white matter astrocyte formation.
Olig2 function is required for white matter astrocyte formation in the spinal cord
A recent fate-mapping study indicated that Olig2-expressing cells
could give rise to astrocytes in the spinal cord
(Masahira et al., 2006). In
light of the profound deficit in white matter astrocyte formation in the brain
of Olig2-ablated mice (CkoG) mediated through
hGFAP-Cre, we examined the spinal cord from these mutants to determine whether
white matter astrocytes in this caudal region of the CNS are affected in the
absence of Olig2. Strong Gfap expression was observed in the white
matter region of the wild-type spinal cord at P14, as was expression of the
other astrocyte markers, S100ß and GS
(Fig. 5). By contrast, in the
spinal white matter of Olig2 mutants, we observed marked
downregulation not only of Gfap mRNA, but also of protein expression
(Fig. 5B,E,F), despite
relatively normal GFAP expression in the gray matter of the spinal cord. In
Olig2-mutant mice, the number of cells expressing S100ß and GS
in the white matter of the spinal cord was markedly reduced
(Fig. 5I-J,M-N), representing
approximately 18% and 25% of that in the controls, respectively
(Fig. 5O). Thus, the reduction
of astrocyte marker expression in the spinal white matter of the
Olig2 mutant suggests that Olig2 is required for the formation of
white matter astrocyte subpopulations in the spinal cord.
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).
The number of GS- cells in the superficial layers of the
Olig2-ablated cortex was comparable to the control
(Fig. 6C,D,J), suggesting that
there is no significant alteration in the extent of cortical astrocyte
formation in the absence of Olig2. Intriguingly, GS+ cells in the
Olig2-ablated cortex became GFAP+, in contrast to the low
level or absence of GFAP expression in the control
(Fig. 6F compared with E).
Similarly, when examining the expression of another astrocyte marker,
S100ß, which co-expresses with Olig2 in the superficial cortical layers
(Fig. 6G), we observed that a
population of S100ß cells acquired a high level of GFAP expression in the
Olig2-ablated cortex, but not in the control
(Fig. 6I compared with H). As
in the white matter, a reduction in S100ß+ cells was observed
in the gray matter (Fig. 6J);
however, this is likely to result from the loss of a subset of
S100ß+ oligodendroglial lineage cells
(Deloulme et al., 2004) in the
Olig2 mutant (Yue et al.,
2006).
To further determine if there is an alteration in astrocyte proliferation,
we examined the cortex of Olig2 mutant (CkoG)
mice at perinatal stage P14 for BrdU incorporation and expression of the
proliferation marker Ki67 (Maeda et al.,
2001; Torp, 2002).
In the Olig2 mutant cortex, the number of BrdU+
proliferating cells at early postnatal stages P7 and P14 after a 4-hour pulse
of BrdU was similar to the control, despite the presence of excessive
GFAP+ cells (Fig. 6L compared
with K; Fig. 6N).
Similarly, these cortical GFAP+ cells did not express Ki67
(Fig. 6M), suggesting that the
high GFAP-expressing cells in the cortex were not in the cycling state at
perinatal stages. In addition, the Ki67 and BrdU labeling suggested that there
was no significant alteration of proliferation among SVZ progenitor cells
(Fig. 6O,P). Together with a
normal number of cortical astrocytes, an absence of local proliferation of
excessive GFAP+ cells suggests that GFAP is upregulated in
astrocytes of the superficial layers in the Olig2-ablated cortex.
Absence of detectable oligodendrocyte-to-astrocyte conversion in the Olig2-ablated cortex
Since oligodendroglial (or O2A) precursors isolated from optic nerves can
adopt an astrocytic fate in vitro under certain culture conditions
(Raff et al., 1983), we
examined whether cortical oligodendroglial cells could acquire ectopic GFAP
expression in the absence of Olig2. Although formation of myelinating
oligodendrocytes is virtually abolished, oligodendrocyte precursors (OPCs)
that express PDGFR (Woodruff et
al., 2001) and NG2 (also known as Cspg4 - Mouse Genome
Informatics) (Nishiyama et al.,
1996) are maintained throughout the Olig2-ablated cortex
(Yue et al., 2006). They are
present within the domain of excessive GFAP expression. When double
immunostaining of PDGFR or NG2 with GFAP was performed in the
Olig2-ablated cortex at P14 (Fig.
7A,B), we did not detect GFAP expression in
PDGFR+ OPCs by confocal imaging
(Fig. 7A-A''). Similarly,
expression of NG2 was clearly distinct from that of GFAP
(Fig. 7B-B''). These
observations indicate that OPCs are either not the source of GFAP-expressing
cells or in so becoming, they lose their classic OPC markers.
|
),
in which Cre is expressed in all stages of oligodendrocytes including their
precursors. Olig2 ablation by CNP-Cre activity resulted in severe
cortical myelination deficit (Fig. 7D
compared with C), but without affecting OPC formation or causing
significant cell death (data not shown). There was, however, an absence of
GFAP upregulation in the cortex at P14 and adulthood in this Olig2
mutant (Fig. 7E,F), suggesting
that persistent cortical GFAP expression is not due to oligodendroglial
lineage cells acquiring ectopic GFAP expression in the absence of
Olig2. In addition, although Olig2 ablation by CNP-Cre activity leads to a deficit in oligodendrocyte myelination in the white matter, there was no apparent alteration in white matter astrocyte formation in the mutant mice, as indicated by a comparable number of GFAP+ or GS+ cells in the white matter in the control and Olig2 CkoC mutant mice (Fig. 7H compared with G). This suggests that the myelination deficit that resulted from Olig2 ablation in committed oligodendrocyte lineage cells does not lead indirectly to a defect in white matter astrocyte formation.
Absence of significant neuronal deficit in the Olig2-ablated cortex
In view of the neurological abnormalities in Olig2-ablated mutants
(Yue et al., 2006), we
examined the effects of Olig2 deletion on cortical neurogenesis, as
reactive astrocytes exhibiting elevated GFAP expression can be induced by
abnormal neuronal formation and function. For cortical neurons in the
Olig2-ablated cortex, cortical lamination and the expression of the
pan-neuronal marker NeuN (also known as Neuna60 - Mouse Genome Informatics)
were comparable with that of control mice
(Fig. 8A,B). Since multipotent
Olig2+ progenitor cells are capable of generating certain
neuronal populations, including GABAergic interneurons, in the developing
cortex (Furusho et al., 2006;
He et al., 2001), we further
examined interneuron subtype development in the Olig2- ablated
cortex. Immunoreactivity for the neurotransmitter GABA, reflecting GABAergic
interneurons and subcortical afferents, appeared to be normal in the
Olig2 mutant (Fig.
8C,D). In addition, we were unable to detect robust changes in
calbindin-expressing interneurons, which are present in superficial layers of
cortex within the domain of GFAP upregulation
(Fig. 8C,D). Furthermore, there
is a lack of marked apoptosis in the cortex of Olig2 mutant mice as
assessed by TUNEL and caspase 3 immunoreactivity (data not shown). Thus, our
observations indicate that Olig2 ablation does not obviously alter
cortical neurogenesis.
|
;
Zhu et al., 2001). The
synapsin I promoter drives Cre recombinase expression in early-postmitotic
neurons as early as embryonic day (E) 12.5, the onset of cortical neurogenesis
(Luikart et al., 2005;
Zhu et al., 2001). Ablation of
Olig2 in early-postmitotic neuronal cells directed by Syn1-Cre did
not lead to GFAP upregulation or alter the number of NeuN+ neurons
in the Olig2 mutant (CkoS) cortex
(Fig. 8F compared with E).
Furthermore, when examining axonal proteins such as neurofilamin 200 (NF200;
also known as Nefh - Mouse Genome Informatics), Tau-1 (Ifne1 - Mouse Genome
Informatics) and Gap43, in the cortex of wild-type and Olig2-ablated
CkoG mice (Fig.
8G-N), we observed normal formation of axonal filaments in
Olig2-ablated brains. Thus, there is an absence of significant axonal
protein deficit in both the gray and white matter of CkoG
mice as compared with control mice.
A subset of cortical astrocytes with sustained GFAP upregulation is derived from Olig2-ablated cells
Since Olig1 has been shown to be co-expressed with Olig2
in the same cells (Zhou et al.,
2000), we examined Olig1 expression in the
Olig2-ablated cortex. Olig1+ cells were present
throughout the gray matter and in numbers comparable to control mice at
perinatal stage P8 (Fig. 9A,B).
At P14, however, Olig1 expression in the superficial cortical layers
was downregulated compared with the control
(Fig. 9D compared with C),
coincident with the appearance of Gfap upregulation
(Fig. 9F compared with E).
Intriguingly, Olig1 expression was maintained in the deep cortical
layers (Fig. 9D,F), but
markedly reduced in the white matter (Fig.
9B,D, arrowheads). This reciprocal pattern of Gfap and
Olig1 expression in the cortex was confirmed by double in situ
hybridization labeling (Fig.
9F). These observations suggest that the absence of Olig2
affects the expression of Olig1, either directly or indirectly, and
that Olig1 may cooperate with Olig2 in regulating cortical astrocyte
development.
|
). Cre-mediated loxP site excision simultaneously
leads to Olig2 ablation and activates YFP expression
(Fig. 9G). Indeed, in the
cortex of triple-transgenic mice (Olig2Cko;hGFAPCre;RosaYFP) at P14,
GFAP expression was observed in YFP-expressing cells, indicating that cortical
astrocytes with GFAP upregulation or high GFAP expression are derived from
Olig2-ablated cells exhibiting YFP reporter expression
(Fig. 9H). Furthermore, to map
the fate of Olig2-ablated cells in the cortex, we examined expression
of the Olig2 mRNA 3' untranslated region (3' UTR) outside
the floxed Olig2 coding sequence
(Fig. 9G). Expression of the
Olig2-3' UTR, transcribed from the Olig2
promoter, represents the cells in which endogenous Olig2 is normally
expressed. In the Olig2 mutant cortex at P14, essentially all
Olig2-3' UTR+ cells expressed Gfap
in the superficial cortical layers but not in deep layers
(Fig. 9I,J), suggesting that
this population of cortical astrocytes with Gfap upregulation is
derived from presumptive Olig2-expressing cells. We observed that not
all Gfap+ cells express Olig2-3'
UTR. This might be due to the downregulation of the Olig2
promoter activity in mature astrocytes
(Gabay et al., 2003).
Alternatively, it is possible that a subset of GFAP+ cortical
astrocytes such as those being generated ventrally would never have expressed
Olig2 during their development. Nonetheless, co-expression of GFAP
with Olig2-3' UTR and the YFP reporter in the
Olig2-ablated cortex suggests a cell-autonomous function of
Olig2 in cortical astrocyte development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Reichenbach and Wolburg, 2005;
Walz, 2000). At present, the
regulatory mechanisms behind gray and white matter astrocyte development are
not fully understood. With tissue-, cell type-, and temporal-specific in vivo
mutagenesis, our present studies unveil a novel and crucial role for Olig2 in
white matter astrocyte formation, about which very little is known, while
illuminating divergent transcriptional regulatory mechanisms and developmental
sources for gray and white matter astrocytes in the developing brain.
Cell-autonomous function of Olig2 in cortical astrocyte development
Previous studies indicate that Olig2 is initially expressed in a
specialized domain (pMN) of the embryonic ventral spinal cord that gives rise
to oligodendrocytes and motoneurons (Lu et
al., 2000; Takebayashi et al.,
2000; Zhou et al.,
2000). In the adult brain, Olig2 is colocalized with markers for
oligodendrocytes but not astrocytes, suggesting that Olig2 is not expressed in
mature astrocytes of the cortex in adulthood
(Lu et al., 2000;
Zhou et al., 2000). Although
most astrocytes do not express a detectable level of GFAP in the adult cortex,
GFAP expression can be detected in a population of immature cortical
astrocytes at early postnatal stages. In the present study, however, we found
that immature GFAP+ astrocytes in the developing cerebral gray and
white matter at early neonatal stages express a high level of Olig2. Olig2
expression subsequently decreases in astrocytes beginning at the perinatal
stage and is completely absent from mature astrocytes at adulthood. Thus,
Olig2 appears to be expressed transiently in developing immature astrocytes at
early postnatal stages but is downregulated progressively as astrocytes mature
in the cortex (Fig. 9K).
In the absence of Olig2, astrocyte formation in the cortex is severely compromised in the white matter, whereas there is a sustained upregulation of GFAP in a population of astrocytes in the gray matter (Fig. 9K). Together with Olig2 expression in immature developing astrocytes and their progenitors in the SVZ in the developing brain, our fate-mapping study further indicates that a population of GFAP+ cortical astrocytes is derived from Olig2-ablated cells expressing the Olig2-3' UTR. Thus, these observations suggest a cell-autonomous function of Olig2 in cortical astrocyte development and that abnormal astrocyte development is, at least in part, attributed to the direct loss of Olig2 function in developing astrocytes and their progenitors.
|
;
Gabay et al., 2003). Our in
vivo mutagenesis analysis, however, indicates that the function of Olig2 in
astrocyte differentiation does not appear to be uniform, but rather
region-specific within the brain. In the gray matter, Olig2 ablation
appears to alleviate likely GFAP repression in a population of presumptive
Olig2+ cortical astrocytes and leads to persistent GFAP
expression throughout adulthood (Fig.
9K). Abnormal GFAP upregulation in developing astrocytes of the
Olig2 mutant cortex is consistent with a GFAP repression function of
Olig2 suggested by in vitro studies. By contrast, our data indicate that Olig2
function is required for astrocyte formation in the white matter of the brain
and the spinal cord. The mechanisms underlying the deficit of white matter
astrocytes in Olig2 mutants are not entirely clear at present. It is
possible that the astrocytes originally destined for white matter somehow end
up in the cortex. This will be resolved in future cell lineage tracing
studies. The requirement for Olig2 in white matter astrocyte
formation is unlikely to be due to a loss of neural progenitor/stem cells
because GFAP+ precursor cells in the SVZ of Olig2 mutants
are maintained at a comparable level to the control. In addition, we do not
observe any significant alteration in the number of proliferative cells
(Ki67+ or BrdU+) or in cell death in the cortex and the
white matter of Olig2 mutants at early postnatal stages, although
this does not exclude the possibility of astrocyte precursor proliferation
deficit, which might be obscured amidst a significant number of proliferative
OPCs and hampered by the lack of well-defined astrocyte precursor markers.
Temporally specific ablation suggests that Olig2 is required for the
differentiation of neural progenitor cells or developing immature astrocyte
precursors at neonatal stages into mature astrocytes in the cerebral white
matter. Alternatively, hypomyelinated axons may create an environment
incompatible with white matter astrocyte formation as there is a myelination
deficit in the white matter of CkoG mice
(Yue et al., 2006). However,
we do not observe significant, if any, deficit in axonal protein expression or
axonal degeneration in the white matter of CkoG mice. In
addition, dysmyelination in the white matter caused by CNP-Cre-mediated
Olig2 ablation in oligodendrocyte lineage cells including
oligodendrocyte progenitors does not cause GFAP upregulation or alter
astrocyte formation, indicating that hypomyelination does not preclude
formation of astrocytes and their presence in the white matter. Thus,
hypomyelination alone is insufficient to account for the deficit in white
matter astrocyte formation. Conversely, given that Olig2 is required
for white matter astrocyte formation, there remains the possibility that
oligodendrocyte deficit in the Olig2 mutant might be caused in part
by the loss of white matter astrocytes, which are normally generated prior to
oligodendrocytes. This possibility will be addressed further in the future by
examining oligodendrocyte formation in the animals with astrocyte-specific
deficits. Nonetheless, because Olig2 expression is observed in a population of
developing GFAP+ white matter astrocytes, the differentiation
deficit of white matter astrocytes is, at least in part, attributed to the
loss of Olig2 function in developing astrocytes and their progenitors.
|
). This is
consistent with observations in the embryonic spinal cord where increased GFAP
expression was only observed with both Olig1- and Olig2-null
mutations (Zhou and Anderson,
2002). Therefore, our observations indicate that gray and white
matter astrocytes differ not only in their spatial locations and morphologies
but also in their transcriptional regulation, suggesting that astrocytes are
not created equally in the developing CNS.
Temporally specific Olig2 ablation reveals diverse developmental sources for cortical gray and white matter astrocyte subpopulations
Cortical astrocytes form from at least two different origins - namely,
radial glial cells at early embryonic stages and SVZ progenitors at early
postnatal stages (Goldman,
2004). Olig2 ablation by inducible Cre activity at early
postnatal stages leads to severe astrocyte differentiation deficit
specifically in the white matter. This suggests that postnatal progenitor
cells, which are likely to reside in the SVZ and the developing white matter,
contribute significantly to the formation of white matter astrocytes in the
developing brain. This observation is in keeping with the inhibitory effects
of Olig2 on astrocyte differentiation when a dominant-interfering
form of Olig2 was introduced into SVZ progenitors at postnatal stages
(Marshall et al., 2005).
However, Marshall et al. (Marshall et al.,
2005) provided evidence that Olig2 function is required
for cortical gray matter astrocyte formation, in contrast to our present study
showing that Olig2 is required for white matter astrocyte generation.
The reasons for this discrepancy are unclear. It is possible that the effects
of genetic Olig2 ablation might differ from those of the
dominant-interfering form of Olig2, or that the timing and cell types where
Olig2 function is perturbed diverge between these two approaches.
Interestingly, postnatal ablation of Olig2 does not lead to GFAP upregulation in the gray matter. By contrast, Olig2 ablation with hGFAP-Cre activity, which is present in cortical progenitors at both early embryonic and postnatal stages, results in not only a defect in white matter astrocyte formation, but also in GFAP upregulation in a subpopulation of cortical astrocytes. By comparison, region-specific alteration of astrocyte subpopulations suggests that there might be different sources for cortical astrocyte development, with a major contribution of early embryonic cortical progenitors to gray matter astrocytes and a significant contribution of postnatal progenitor cells to white matter astrocytes.
|
), ventrally derived astrocytes may migrate and
contribute to astrocyte formation in the white matter. Together, our
observations suggest that diverse developmental sources for gray and white
matter astrocytes might exist in the developing CNS.
Olig2 function in cortical neurogenesis
Although there is a deficit in motoneuron formation in the spinal cord of
Olig2-null mice, Olig2 ablation in cortical progenitor cells
and early-postmitotic neurons does not lead to a significant abnormality in
cortical neurogenesis. The lack of any overt alteration in cortical neuronal
formation is rather surprising because recent studies indicate that expression
of dominant-negative forms of Olig2 leads to an increase in neurogenesis
during development (Marshall et al.,
2005) and to new neuron formation in response to injury
(Buffo et al., 2005). One
potential explanation for this discrepancy is that Olig2 ablation in
the cortical progenitor cells might lead to an increase in neuronal formation,
with newly formed neurons subsequently eliminated through programmed cell
death. We do not observe, however, any appreciable increase in cell death or
the alteration of neural patterning in the cortex. Alternately, the
dominant-negative forms of Olig2 used could potentially interact with other
transcriptional regulators such as neural bHLH factors in addition to
inhibiting Olig2, thereby altering cortical neurogenesis. Nonetheless, the
lack of any obvious neuronal abnormality in the Olig2-ablated cortex
suggests that elimination of Olig2 function does not significantly alter
neurogenesis in the cortex, at least under the present ablation conditions, in
contrast to motoneuron formation in the developing spinal cord where Olig2
plays an essential role.
Distinct and convergent developmental requirements for cortical oligodendroglial and astroglial lineage development
Since Olig2 negatively regulates astrocyte differentiation in neural
progenitor cells in vitro, this raises the possibility that Olig2
might regulate an intrinsic fate switch for glial subtypes. Intriguingly, in
the Olig2-ablated cortex, OPCs present within the domain of ectopic
GFAP expression do not acquire GFAP expression. Furthermore, Olig2
ablation in oligodendrocyte lineage cells with the oligodendrocyte-specific
CNP-Cre line does not lead to upregulation of GFAP in the cortex. Together,
these data suggest that committed oligodendroglial lineage cells in the cortex
do not adopt an astrocytic fate in the absence of Olig2, although
they fail to further differentiate into mature myelinating oligodendrocytes
(Yue et al., 2006).
Conversely, Olig2 ablation leads to a decrease in both
oligodendrocytes and astrocytes in the white matter, suggesting that there is
a common requirement for Olig2 for glial subtype differentiation in
the white matter tract. Intriguingly, OPCs isolated from the neonatal rat
optic nerve tract can differentiate into fibrous astrocytes if cultured under
certain conditions (Raff et al.,
1983). The fact that Olig2 is required for both
oligodendrocyte and white matter astrocyte differentiation suggests that
oligodendrocytes and astrocytes in the white matter might develop from the
same source, although further evidence for this cell lineage connection needs
to be obtained. Since astrocytes in the gray matter are formed in the absence
of Olig2, our results suggest that the development of astrocytes in
the gray and white matter are regulated by distinct developmental mechanisms,
although there might be a convergent regulatory mechanism between
oligodendrocytes and astrocytes in the white matter during brain development.
Thus, spatiotemporal-specific Olig2 ablation reveals divergent
regulatory mechanisms for the development of gray and white astrocyte
subpopulations and highlight important roles of Olig2 in glial
subtype development.
|
ACKNOWLEDGMENTS |
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REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Buffo, A., Vosko, M. R., Erturk, D., Hamann, G. F., Jucker, M.,
Rowitch, D. and Gotz, M. (2005). Expression pattern of the
transcription factor Olig2 in response to brain injuries: Implications for
neuronal repair. Proc. Natl. Acad. Sci. USA
102,18183
-18188.
Connor, J. R. and Berkowitz, E. M. (1985). A demonstration of glial filament distribution in astrocytes isolated from rat cerebral cortex. Neuroscience 16, 33-44.[CrossRef][Medline]
Deloulme, J. C., Raponi, E., Gentil, B. J., Bertacchi, N., Marks, A., Labourdette, G. and Baudier, J. (2004). Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation. Mol. Cell. Neurosci. 27,453 -465.[CrossRef][Medline]
Forster, E., Zhao, S. and Frotscher, M. (2006). Laminating the hippocampus. Nat. Rev. Neurosci. 7, 259-267.[CrossRef][Medline]
Fukuda, S., Kondo, T., Takebayashi, H. and Taga, T. (2004). Negative regulatory effect of an oligodendrocytic bHLH factor OLIG2 on the astrocytic differentiation pathway. Cell Death Differ. 11,196 -202.[CrossRef][Medline]
Furusho, M., Ono, K., Takebayashi, H., Masahira, N., Kagawa, T., Ikeda, K. and Ikenaka, K. (2006). Involvement of the Olig2 transcription factor in cholinergic neuron development of the basal forebrain. Dev. Biol. 293,348 -357.[CrossRef][Medline]
Gabay, L., Lowell, S., Rubin, L. L. and Anderson, D. J. (2003). Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron 40,485 -499.[CrossRef][Medline]
Goldman, J. E. (2004). Astrocyte lineage. In Myelin Biology and Disorders, vol.1 (ed. R. A. Lazzarini), pp.311 -328. New York: Elsevier Academic Press.
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244,305 -318.[CrossRef][Medline]
He, W., Ingraham, C., Rising, L., Goderie, S. and Temple, S.
(2001). Multipotent stem cells from the mouse basal forebrain
contribute GABAergic neurons and oligodendrocytes to the cerebral cortex
during embryogenesis. J. Neurosci.
21,8854
-8862.
Kimelberg, H. K. (2004). The problem of astrocyte identity. Neurochem. Int. 45,191 -202.[CrossRef][Medline]
Lappe-Siefke, C., Goebbels, S., Gravel, M., Nicksch, E., Lee, J., Braun, P. E., Griffiths, I. R. and Nave, K. A. (2003). Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination. Nat. Genet. 33,366 -374.[CrossRef][Medline]
Lu, Q. R., Yuk, D., Alberta, J. A., Zhu, Z., Pawlitzky, I., Chan, J., McMahon, A. P., Stiles, C. D. and Rowitch, D. H. (2000). Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system. Neuron 25,317 -329.[CrossRef][Medline]
Lu, Q. R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C. D. and Rowitch, D. H. (2002). Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection. Cell 109,75 -86.[CrossRef][Medline]
Luikart, B. W., Nef, S., Virmani, T., Lush, M. E., Liu, Y.,
Kavalali, E. T. and Parada, L. F. (2005). TrkB has a
cell-autonomous role in the establishment of hippocampal Schaffer collateral
synapses. J. Neurosci.
25,3774
-3786.
Maeda, Y., Solanky, M., Menonna, J., Chapin, J., Li, W. and Dowling, P. (2001). Platelet-derived growth factor-alpha receptor-positive oligodendroglia are frequent in multiple sclerosis lesions. Ann. Neurol. 49,776 -785.[CrossRef][Medline]
Malatesta, P., Hack, M. A., Hartfuss, E., Kettenmann, H., Klinkert, W., Kirchhoff, F. and Gotz, M. (2003). Neuronal or glial progeny: regional differences in radial glia fate. Neuron 37,751 -764.[CrossRef][Medline]
Marshall, C. A., Novitch, B. G. and Goldman, J. E.
(2005). Olig2 directs astrocyte and oligodendrocyte formation in
postnatal subventricular zone cells. J. Neurosci.
25,7289
-7298.
Masahira, N., Takebayashi, H., Ono, K., Watanabe, K., Ding, L., Furusho, M., Ogawa, Y., Nabeshima, Y., Alvarez-Buylla, A., Shimizu, K. et al. (2006). Olig2-positive progenitors in the embryonic spinal cord give rise not only to motoneurons and oligodendrocytes, but also to a subset of astrocytes and ependymal cells. Dev. Biol. 293,358 -369.[CrossRef][Medline]
Mehler, M. F. (2002). Mechanisms regulating lineage diversity during mammalian cerebral cortical neurogenesis and gliogenesis. Results Probl. Cell Differ. 39, 27-52.[Medline]
Miller, R. H. and Raff, M. C. (1984). Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. J. Neurosci. 4,585 -592.[Abstract]
Miller, R. and Reynolds, R. (2004). Oligodendroglial lineage. In Myelin Biology and Disorders, vol. 1 (ed. R. A. Lazzarini), pp. 289-310. New York: Elsevier Academic Press.
Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H. and Stallcup, W. B. (1996). Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain. J. Neurosci. Res. 43,299 -314.[CrossRef][Medline]
Raff, M. C., Miller, R. H. and Noble, M. (1983). A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303,390 -396.[CrossRef][Medline]
Reichenbach, A. and Wolburg, H. (2005). Astrocytes and ependymal glia. In Neuroglia, (ed. H. Kettenmann and B. R. Ransom), pp. 19-35. New York: Oxford University Press.
Rickmann, M. and Wolff, J. R. (1995). S100 immunoreactivity in a subpopulation of oligodendrocytes and Ranvier's nodes of adult rat brain. Neurosci. Lett. 186, 13-16.[CrossRef][Medline]
Ridet, J. L., Malhotra, S. K., Privat, A. and Gage, F. H. (1997). Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 20,570 -577.[CrossRef][Medline]
Sauvageot, C. M. and Stiles, C. D. (2002). Molecular mechanisms controlling cortical gliogenesis. Curr. Opin. Neurobiol. 12,244 -249.[CrossRef][Medline]
Setoguchi, T. and Kondo, T. (2004). Nuclear
export of OLIG2 in neural stem cells is essential for ciliary neurotrophic
factor-induced astrocyte differentiation. J. Cell
Biol. 166,963
-968.
Shu, T. and Richards, L. J. (2001). Cortical
axon guidance by the glial wedge during the development of the corpus
callosum. J. Neurosci.
21,2749
-2758.
Sofroniew, M. V. (2005). Reactive astrocytes in
neural repair and protection. Neuroscientist
11,400
-407.
Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M. and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1,4 .[CrossRef][Medline]
Stanimirovic, D. B., Ball, R., Small, D. L. and Muruganandam, A. (1999). Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro. Int. J. Dev. Neurosci. 17,173 -184.[Medline]
Sun, Y. E., Martinowich, K. and Ge, W. (2003). Making and repairing the mammalian brain - signaling toward neurogenesis and gliogenesis. Semin. Cell. Dev. Biol. 14,161 -168.[CrossRef][Medline]
Takebayashi, H., Yoshida, S., Sugimori, M., Kosako, H., Kominami, R., Nakafuku, M. and Nabeshima, Y. (2000). Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3. Mech. Dev. 99,143 -148.[CrossRef][Medline]
Takebayashi, H., Nabeshima, Y., Yoshida, S., Chisaka, O. and Ikenaka, K. (2002). The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages. Curr. Biol. 12,1157 -1163.[CrossRef][Medline]
Temple, S. (2001). The development of neural stem cells. Nature 414,112 -117.[CrossRef][Medline]
Torp, S. H. (2002). Diagnostic and prognostic role of Ki67 immunostaining in human astrocytomas using four different antibodies. Clin. Neuropathol. 21,252 -257.[Medline]
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S. and
Barres, B. A. (2001). Control of synapse number by glia.
Science 291,657
-661.
Walz, W. (2000). Controversy surrounding the existence of discrete functional classes of astrocytes in adult gray matter. Glia 31,95 -103.[CrossRef][Medline]
Woodruff, R. H., Tekki-Kessaris, N., Stiles, C. D., Rowitch, D. H. and Richardson, W. D. (2001). Oligodendrocyte development in the spinal cord and telencephalon: common themes and new perspectives. Int. J. Dev. Neurosci. 19,379 -385.[CrossRef][Medline]
Xin, M., Yue, T., Ma, Z., Wu, F. F., Gow, A. and Lu, Q. R.
(2005). Myelinogenesis and axonal recognition by oligodendrocytes
in brain are uncoupled in Olig1-null mice. J.
Neurosci. 25,1354
-1365.
Yue, T., Xian, K., Hurlock, E., Xin, M., Kernie, S. G., Parada,
L. F. and Lu, Q. R. (2006). A critical role for dorsal
progenitors in cortical myelination. J. Neurosci.
26,1275
-1280.
Zhou, Q. and Anderson, D. J. (2002). The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell 109,61 -73.[CrossRef][Medline]
Zhou, Q., Wang, S. and Anderson, D. J. (2000). Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25,331 -343.[CrossRef][Medline]
Zhu, Y., Romero, M. I., Ghosh, P., Ye, Z., Charnay, P., Rushing,
E. J., Marth, J. D. and Parada, L. F. (2001). Ablation of NF1
function in neurons induces abnormal development of cerebral cortex and
reactive gliosis in the brain. Genes Dev.
15,859
-876.
Zhu, Y., Harada, T., Liu, L., Lush, M. E., Guignard, F., Harada,
C., Burns, D. K., Bajenaru, M. L., Gutmann, D. H. and Parada, L. F.
(2005). Inactivation of NF1 in CNS causes increased glial
progenitor proliferation and optic glioma formation.
Development 132,5577
-5588.
Zhuo, L., Theis, M., Alvarez-Maya, I., Brenner, M., Willecke, K. and Messing, A. (2001). hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo. Genesis 31,85 -94.[CrossRef][Medline]
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