1 Division of Molecular Neurobiology, National Institute for Physiological
Sciences, Okazaki, Aichi 444-8585, Japan
2 Department of Developmental Genetics, National Institute of Genetics, Mishima,
Shizuoka 411-8540, Japan
* Author for correspondence (e-mail: ikenaka{at}nips.ac.jp)
Accepted 27 August 2003
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SUMMARY |
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Key words: glial cells missing (gcm), Glial development, Astrocyte, Retrovirus
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Introduction |
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Two mammalian gcm homologs (Gcm1 and Gcm2,
previously known as Gcma and Gcmb) have been identified in
mice, rats and humans (Akiyama et al.,
1996; Altshuller et al.,
1996
; Kammerer et al.,
1999
; Kanemura et al.,
1999
; Kim et al.,
1998
). Sequence homology between Drosophila and mammalian
Gcm proteins is restricted to the N-terminal region, which contains the
DNA-binding domain. In agreement, all Gcms bind the same DNA sequence,
(A/G)CCCGCAT (Akiyama et al.,
1996
; Schreiber et al.,
1998
). When mouse Gcm1 was ectopically expressed in the
Drosophila nervous system, formation of additional glial cells was
observed (Kim et al., 1998
;
Reifegerste et al., 1999
).
This indicates that mouse Gcm1 is functionally similar to Drosophila
Gcm. The sequence conservation and the interchangeable activity initially led
us to predict that mammalian Gcm plays a role in gliogenesis. Contrary to this
expectation, mammalian Gcm genes were expressed in the nervous system at
extremely low levels, detectable only by sensitive RT-PCR
(Altshuller et al., 1996
;
Basyuk et al., 1999
;
Kammerer et al., 1999
;
Kanemura et al., 1999
;
Kim et al., 1998
). The main
sites of Gcm1 and Gcm2 expression are the placenta
(Basyuk et al., 1999
;
Kim et al., 1998
) and
parathyroid glands (Kim et al.,
1998
), respectively. Targeted disruption of the mouse
Gcm1 gene in mice results in a severe defect in labyrinth formation
in the placenta, which leads to embryonic lethality between embryonic day 9.5
and 10 (E9.5-10) (Anson-Cartwright et al.,
2000
; Schreiber et al.,
2000
). No abnormalities were detected in the embryo proper at
least until death. By contrast, mouse Gcm2-targeted mice exhibit a
selective loss of the parathyroid glands, but no abnormalities were reported
in the nervous system (Gunther et al.,
2000
). These findings raise the speculation that mammalian Gcm
genes have a biological role other than in gliogenesis.
In the present study, we have elucidated the function of mammalian Gcm
genes in the central nervous system by employing retrovirus-mediated gene
expression. In developing brain cells, mouse Gcm1 induced astrocyte
cell fate and suppressed neuronal cell fate both in vitro and in vivo.
However, the expression pattern of the mouse Gcm1 gene was distinct from those
observed for the early astrocyte markers, such as GLAST and
brain-lipid-binding protein (BLBP)
(Shibata et al., 1997;
Hartfuss et al., 2001
).
Moreover, brain cultures from mouse Gcm1-deficient mice did not
display a significant reduction in the number of astrocytes. Taken together,
we speculate that mouse Gcm1 may functionally contribute towards the
generation of a minor subpopulation of glial cells.
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Materials and methods |
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Retroviral vectors
The gcm and mouse Gcm cDNAs
(Akiyama et al., 1996;
Hosoya et al., 1995
) were
cloned into a retroviral vector, pTY20E+
(Ikeda et al., 1997
), which
has an internal ribosome entry sequence (IRES) followed by the lacZ
gene. A control vector contains a neor gene instead of a
gcm gene. Retroviral plasmid DNA was transfected into retroviral
packaging cells,
MP34 (Yoshimatsu et
al., 1998
), using LipofectAMINE plus Reagent (Invitrogen).
MP34 was derived from the NIH3T3 cell line and modified to provide
high-titer virus (Yoshimatsu et al.,
1998
). Several days after transfection,
ß-galactosidase-positive cells were collected by fluorescent activated
cell sorter, FACS Vantage (BD Biosciences) using a fluorogenic substrate,
fluorescein di-ß-galactopyranoside according to a method (FDG-FACS)
previously reported (Nolan et al.,
1988
). Fluorescein di-ß-galactopyranoside was synthesized as
described (Ikenaka et al.,
1990
). FACS was repeated until stable transformants were obtained.
Supernatants containing virus were collected and titered on NIH3T3 cells.
Concentration and medium change of viral solutions were performed by
centrifugation at 6000 g at 4°C for 16 hours
(Bowles et al., 1996
). For the
histochemical detection of ß-galactosidase, cultures were fixed with 0.5%
glutaraldehyde and then incubated with 1 mg/ml X-gal
(5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside) solution, including 5
mM K3Fe(CN)6, 5 mM K4Fe(CN)6,
0.01% sodium deoxycholate, 0.02% Nonidet P-40 and 1 mM MgCl2 in PBS
at 37°C overnight. Rabbit polyclonal anti-mouse Gcm1 (peptide antibody
against DVKLPQNVKTTDWFQEWPDS) and anti-mouse Gcm2 (antibody against GST fusion
mouse Gcm2 protein; a gift from Dr Masato Nakafuku, University of Tokyo) were
used to detect the expressed mouse Gcm proteins in each stable transformant.
Retroviral vectors containing alkaline phosphatase
(Ikeda et al., 1997
) were used
as an internal control for culture conditions. For colony formation assays,
NIH3T3 cells (1x104) were seeded onto 3.5 cm dishes and
infected with recombinant virus with 8 µg/ml polybrene (Sigma-Aldrich) the
next day. Five days later, cultures were stained with X-gal and the numbers of
cells in clusters were counted.
Primary cell culture and retrovirus-mediated gene expression
Hemispheres were dissected from ICR mouse embryos (E12.5) and mechanically
triturated in medium by passing through a heat-polished Pasteur pipette.
Dissociated cells were washed with DMEM/F12 medium (DF; Sigma) containing 10%
fetal bovine serum (FBS; EQUITECH-BIO) and plated on eight-well slides coated
with polyethyleneimine (Sigma) at a density of 1x105
cells/cm2. The following day, cells were incubated with
retroviruses (1x106 cfu/cm2) with 1 µg/ml
polybrene for 4 hours, and then the medium was replaced with serum-free DF
medium containing N2 supplement (Invitrogen). Two or three days after
infection, cultures were fixed with 2% paraformaldehyde (PFA) and 0.02%
glutaraldehyde in PBS, and incubated in a X-Gal solution at 37°C for 4-8
hours. Following the X-gal reaction, cells were stained with anti-MAP-2
(Sigma) or anti-GFAP (DAKO) antibodies. For O4 staining, the cells were
incubated with O4 antibody (a gift from Dr Steven E. Pfeiffer, University of
Connecticut) before fixation. The cells were subsequently incubated with
biotinylated goat anti-mouse IgM antibody (Vector Laboratories), followed by
Vectastain ABC-peroxidase detection (Vector Laboratories). For
S100ß+ staining, the culture was fixed with 4% PFA and
incubated with anti-ß-gal (Cappel) and anti-S100ß (Sigma)
antibodies, followed by detection with secondary antibodies conjugated to
Alexa488 or Alexa594 (Molecular Probes). Detection of
alkaline phosphatase activity was performed as described previously
(Ikeda et al., 1997
).
Prenatal retrovirus injections and histochemical analysis
Pregnant mice containing E13 embryos were anesthetized with Nembutal
(Abbott), and after midline laparotomy, fetal heads were transilluminated with
a fiber optic source, and the location of the lateral ventricles was
identified. A retroviral vector carrying the lacZ reporter gene was
injected into the lateral ventricle of each embryo. Approximately 1 µl of
retroviral suspension at a titer of 1x109 cfu/ml with 0.05%
Fast Green (Sigma) was injected through a glass capillary. The injected
fetuses were born normally and allowed to survive for up to 2 months. P24 mice
were used for histological analysis. They were perfused with a fixative
solution containing 4% paraformaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 and
0.1 M phosphate buffer (pH 7.4). The brains were removed from the skulls and
washed in PBS containing 1 mM MgCl2 at 4°C three times.
Subsequently, the brains were incubated in a X-gal solution. After the X-gal
reaction, brains were sectioned serially in the coronal plane at 100 µm
using a Vibratome, or at 30 µm using a Cryostat for further staining with
GFAP and MAP2 antibodies.
Analysis of mouse Gcm1 deficient mice
Brain cells from E9.5 mouse Gcm1 mutant mice (T.H. and Y.H,
unpublished) were cultured according to Kitani's method
(Kitani et al., 1991) with
some modifications. Briefly, heads were dissected and incubated with 0.25%
trypsin for 7 minutes at 37°C. After addition of FBS, cells were
triturated in a DF medium supplemented with 10% FBS by pipeting and plated on
a non-coated 3.5 cm dish. After incubation for 4 hours, untouched cells were
collected and cultured again on a non-coated 3.5 cm dish. The following day,
untouched cells were collected and plated on a PEI-coated eight-well glass
slide. Their embryonic bodies were used for PCR genotyping. Primers were:
lacZ, (5'-attaggtccctcgaaggaggttcac-3',
5'-tgagtttatgttccaccgtgcagc-3'); and mouse Gcm1,
(5'-aacgactgactggttccaggagtgg-3',
5'-ggccttgtcacagatggctggcctcag-3'). After 3 or 5 days, cultures
were fixed with 4% PFA and incubated in blocking solution (PBS with 3% normal
goat serum and 0.1% Triton X-100) for 1 hour at room temperature. Cells were
then stained with anti-MAP2, S100ß and GFAP antibodies, followed by
detection with secondary antibodies conjugated with Alexa488 or
Alexa594 (Molecular Probes). Cell nuclei were stained with
4',6-diamidino-2-phenylindole (DAPI).
In situ detection of mouse Gcm1 mRNA in the embryonic
brain
In situ hybridization was performed as previously described
(Kagawa et al., 1994). For
preparation of embryonic brain tissues, perfusion fixation using 4%
paraformaldehyde was performed. After fixation, tissues were embedded in
paraffin wax, sectioned at 8 µm and put on APS-coated slides (Matsunami).
Sections were treated with 0.2% pepsin for 2-3 minutes at 37°C, and
hybridized with 100 ng/ml riboprobe in 50% formamide, 20 mM Tris-HCl (pH 7.5),
600 mM NaCl, 1 mM EDTA, 10% dextran sulfate, 200 µg/ml yeast tRNA,
1xDenhardt's solution, 0.25% SDS at 50°C overnight. The sections
were washed with 2xSSC containing 50% formaldehyde at 50°C for 20
minutes, followed by 0.2xSSC at 50°C for 20 minutes twice.
Digoxigenin (DIG)-labeled riboprobes were synthesized from linearized mouse
Gcm1 plasmids (359 bp fragment of mouse Gcm1 cDNA; position
1260-1619, GenBank Accession Number D88612) using the DIG RNA labeling kit
(Roche). DIG probes were visualized by alkaline phosphatase-conjugated
anti-DIG antibody and NBT/BCIP reaction (Roche). Sense probe was used as a
control.
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Results |
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We speculated that these cellular effects are caused by transactivation of certain genes by Gcm, and thus examined expression of several glial genes by RT-PCR. Among several genes, the expression of S100ß, a Ca2+-binding protein expressed in astrocytes, was highly upregulated in mouse Gcm1-transduced cells (Fig. 2L). Significant induction of S100ß was also observed in mouse Gcm2-transduced cells. Expression of S100ß was minimal in the control-transduced (Fig. 2L) or non-transduced (data not shown) fibroblast cells. Expression and induction of GFAP or PLP mRNAs were not detected in these fibroblast cells. GLAST, which is expressed in the early glial lineage, was highly expressed in non-transduced fibroblast cells and a change in expression level owing to Gcm gene transduction was not evident (data not shown).
Effect of gcm-expression on cultured embryonic brain
cells
In order to determine whether the mouse Gcm genes are involved in glial
cell fate determination in the developing nervous system, we forced the
expression of mouse Gcm1 or mouse Gcm2 in cells cultured
from E12 mouse brains. Following retroviral infection, cells were cultured in
a chemically defined medium for 3 days. The cells were fixed and stained with
X-Gal, followed by staining with neuronal marker MAP2, astrocyte marker GFAP
or oligodendrocyte marker O4. Under these culture conditions from early
embryonic brains, only a small number of cells (<1%) were GFAP+
after 3 days, although many cells (30%) were MAP2+ (data not
shown). An additional 2 days in culture led to the appearance of many
GFAP+ cells in the culture. In control experiments, 3% of the
transduced cells were GFAP+
(Fig. 3A,H), but mouse
Gcm1-expression increased this percentage to more than 30%
(Fig. 3B,H). Conversely, 13% of
the transduced cells were MAP2+ in the control
(Fig. 3D,I), whereas mouse
Gcm1-expression decreased this to 3%
(Fig. 3E,I). These results
suggest that mouse Gcm1 induces the astrocyte lineage while
suppressing the neuronal lineage. However, mouse Gcm2 transduction
exhibited no significant differences in comparison with the control
(Fig. 3C,F). With regard to the
oligodendrocyte lineage, we could not detect O4-positive cells after 3 days
culture (data not shown). An additional 3 days in culture led to the
appearance of O4-positive cells, but most of the transduced cells were still
O4 negative and we were unable to detect any significant effects (data not
shown). Induction of GFAP+ cells by mouse Gcm1 was also
observed in the culture after 2 days (Fig.
3G), when GFAP+ cells seldom exist in the control
cultures. It was noteworthy that the only mouse Gcm1-transduced
X-gal+ cells became GFAP+ and the surrounding cells were
negative (Fig. 3G).
Furthermore, the percentage of GFAP+ cells in transduced cells
after 2 days already reached 25%. This indicates that the induction of
GFAP+ cells by mouse Gcm1 was prompt.
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Although longer cultures of transduced cells were attempted, this proved difficult because of gradual cell death caused by mouse Gcm1 transduction (data not shown). This may be attributed to the misexpression and/or overexpression of mouse Gcm1. The cell death of many cells may exhibit negative influences on other cells in culture. We were afraid that such unfavorable conditions in the mouse Gcm gene-transduced culture promoted astrocyte differentiation. To exclude this possibility, we performed mouse Gcm1-lacZ viral infections together with alkaline phosphatase (ALK) virus as an internal control (Fig. 3O,P). No significant differences were observed in the appearance of GFAP+ cells in ALK+ cells in culture between the control and mouse Gcm1 experiment (Fig. 3P). This indicated that the increase of GFAP+ cells was directly induced by mouse Gcm1 expression, not by detrimental culture conditions after massive cell death.
Retrovirus-mediated expression of mouse Gcm1 in vivo
To address whether mouse Gcm1 transduction induces glial lineage
in vivo, we performed in utero injection of retroviruses into developing mouse
brains. Concentrated viral stocks (1x109 cfu/ml) of control
and mouse Gcm1 viruses were prepared and injected into the lateral
ventricle of E13 brains using glass capillaries. The brains were then fixed at
P24, stained with X-gal, and sectioned serially in a coronal plane at 100
µl using a Vibratome. Fig. 4
shows the section of forebrain injected with control (CT) viruses where
morphologies of X-gal+ cells were suggestive of neurons and
astrocytes. Neuron-like cells seemed to exhibit a small clear cell body while
astrocyte-like cells have a large obscure cell body. To confirm this
classification, the cells were double-labeled with X-gal and a neuronal
marker, NeuN (Fig. 4C,D), or an
astrocyte marker, GFAP (Fig.
4E,F) antibody. Among 73 X-gal+ cells morphologically
classified as astrocytes, 72 cells (98.6%) were GFAP+
(Fig. 4E,F), and none (0%) were
MAP2+ (Fig. 4D). As
reported previously, astrocytes in the white matter or near pia mater
exhibited strong immunoreactivity for GFAP
(Fig. 4E), while gray matter
astrocytes exhibited much weaker staining
(Fig. 4F), yet they were
identifiable under our staining conditions. By contrast, among 147
X-gal+ cells morphologically classified as neurons, 109 cells
(74.1%) were NeuN+ cells (Fig.
4C), and none (0%) were GFAP+. Based on this
classification, we scored cell types in the neocortex of P24 brains infected
with control or mouse Gcm1 virus at E13. mouse Gcm1
expression led to a significant increase in the number of astrocytes, and
decrease in the number of neurons, in comparison with the control
(Fig. 2G). Expression of mouse
Gcm1 was also shown to effectively promote the generation of
astrocyte lineage cells in vivo.
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Discussion |
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The present study provides several important insights into the function of
mammalian Gcm in the nervous system. First, we showed that mouse Gcm1
and mouse Gcm2 are expressed in the embryonic brain throughout
development by real time PCR. Next, forced expression studies using a
retroviral vector indicated that mouse Gcm1 indeed promotes astrocyte
lineage and suppresses neuronal lineage in cultured cells from E12 mouse
brains. This induction was so prompt that GFAP+ cells appeared only
2 days after infection. The induction of astrocytes by mouse Gcm1 was
also detected by in utero injection of the retroviral vector into embryonic
brains. Previous reports have demonstrated that ectopic expression of mouse
Gcm1 and mouse Gcm2 in the mouse retina failed to cause
neuron-to-glia transformation (Hojo et al.,
2000). This discrepancy with our results may be explained by the
differences in Müller cells in the retina and in astrocytes referred to
in these studies. Drosophila has two types of glia, longitudinal and
midline glia, but gcm is involved only in longitudinal glial
differentiation. Mammalian astrocytes exhibit a large heterogeneity differing
in morphology, distribution, molecule types expressed, function and cell
lineage, including gray matter astrocytes, white matter astrocytes,
Müller glia in the retina, Bergman glia in the cerebellum and radial
glia. Similar to Drosophila gcm, differences in Gcm involvement may
occur among these cells.
Our in vivo and in vitro studies have demonstrated that mouse Gcm1
has the capacity to induce astrocyte lineage cells, but ablation of the mouse
Gcm1 gene did not cause a significant decrease in GFAP+
cells in cultures from mutant brains. Furthermore, while in situ expression of
mouse Gcm1 was detectable in embryonic brains, it did not coincide
with the expression of well-known glial lineage markers. These discrepancies
strongly suggest that mouse Gcm1-expressing cells are a subpopulation
of glial cells, distinct from the major astrocyte cell type generated around
the P0 in the cortex. Accordingly, data from recent experiments employing
retroviral labeling with an ultrasonic injection system have demonstrated a
population of early glial lineage existing at E9.5
(McCarthy et al., 2001). This
indicates that the specification of some glial cell populations occurs much
earlier than believed previously. Our in situ hybridization data revealed many
mouse Gcm1 signals dispersed in the ganglionic eminence and thalamus.
The instability of mouse Gcm1 signals in the brain, however, makes
signal detection largely dependent on the conditions of tissue fixation. Our
RT-PCR analysis demonstrated that mouse Gcm1 is expressed at higher
levels at E12 than at E14, yet the detectable in situ hybridization signals at
E12 were not stronger (data not shown). This might be due to the omission of
heart-penetrated perfusion of fixative at E12. Quick fixation may be necessary
to avoid degradation of mouse Gcm1 messages.
Mammalian Gcm exhibits DNA-binding specificity similar to
Drosophila Gcm (Akiyama et al.,
1996; Schreiber et al.,
1998
). One of the native targets for Drosophila Gcm is
the repo gene, which contains eleven Gcm-binding sites in its
upstream region (Akiyama et al.,
1996
). Gcm-binding sites were also found in trophoblast-specific
element 2 (TSE2), which is a cis-element that functions as a placenta-specific
enhancer of the human aromatase gene
(Yamada et al., 1999
). In the
present study, we demonstrated that ectopic expression of mouse Gcm1
in mouse fibroblasts led to the induction of the gene encoding the
astrocyte-specific Ca2+-binding protein, S100ß. Analysis of
the promoter region in the mouse S100ß gene revealed the presence of six
Gcm-binding-like sequences. Further analysis is necessary to elucidate the
regulation of S100ß promoter by mouse Gcm1.
It is noteworthy that mouse Gcm2 and mouse Gcm1 exhibited
similar effects on fibroblasts, but only mouse Gcm1 induced glial
lineage in brain cells. This suggests that mouse Gcm2 is a transcriptional
modulator, but is not involved in glial differentiation. However, it has been
reported that mouse Gcm2 contains a unique labile domain that suppresses its
transcriptional activity by degradation
(Tuerk et al., 2000). Thus, it
is possible that although mouse Gcm2 has the potential to induce astrocytes,
its activities are suppressed under normal circumstances. This raises the
speculation that the mouse Gcm1 defect is compensated by mouse
Gcm2 in our experiments. Further analysis using double knockout mice
is necessary to explore this possibility.
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ACKNOWLEDGMENTS |
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