1 Institut Pasteur, Unité de Neurovirologie et
Régénération du Système Nerveux, 75015 Paris,
France
2 Département de Morphologie, Centre Médical Universitaire, 1211
Geneva, Switzerland
* Author for correspondence (e-mail: bruzzone{at}pasteur.fr )
Accepted 11 June 2002
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Summary |
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Key words: Astrocyte, CNS, Connexin, Gap junction, Neuron, Oligodendrocyte
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Introduction |
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Connexins are expressed in the three main cell types of the central nervous
system (CNS), that is, astrocytes, oligodendrocytes and neurons. Increasing
evidence suggests that connexins regulate different aspects of neuronal
networks and neuron-glia interactions, in both the developing and mature brain
(for a review, see Dermietzel and Spray,
1998). This would require the establishment of communication
compartments that isolate groups of coupled cells engaged in a coordinated
activity from other populations that participate in distinct processes. Such a
dynamic regulation of cell-cell communication seems particularly well suited
to respond to the instructive cues operating during CNS development
(Kandler and Katz, 1995
;
Nadarajah et al., 1997
;
Naus and Bani-Yaghoub, 1998
).
Thus, in vivo studies have shown that, in the developing neocortex,
neuroblasts and proliferating cells located in the ventricular zone are
coupled, whereas differentiating or migrating neurons are not
(LoTurco and Kriegstein, 1991
;
Bittman et al., 1997
). Evidence
for coupling territories has also been obtained in other areas of
neurogenesis, such as the subventricular zone and rostral migratory stream
(Miragall et al., 1997
;
Menezes et al., 2000
).
Furthermore, there is an inverse correlation between the expression of
connexin43 (Cx43) and Cx26, and cell proliferation
(Miragall et al., 1997
;
Bittman and LoTurco, 1999
),
suggesting that coupling and cell cycle of neural progenitors may be
interdependent.
Studies in vitro have shown that Cx43 expression and coupling are
downregulated as differentiation proceeds in either immortalized rodent
neuroblasts (Rozental et al.,
1998) or cell lines derived from rat peripheral neurotumoral cells
(Donahue at al., 1998
). In a
human teratocarcinoma cell line consisting of precursors able to differentiate
into post-mitotic neurons, Cx43 protein expression is decreased after neuronal
differentiation, and a pharmacological blockade of gap junctions antagonizes
the acquisition of the neuronal phenotype
(Bani-Yaghoub et al., 1999a
;
Bani-Yaghoub et al., 1999b
).
Taken together, these observations strongly suggest that, on the one hand,
Cx43 plays a role in mediating intercellular coupling during proliferation of
neural precursors and, on the other hand, a downregulation of coupling and
Cx43 expression occurs with cell differentiation.
To gain insight into the role of connexins and junctional communication
during the early steps of CNS differentiation, we have taken advantage of a
primary culture of neural progenitor cells
(Reynolds and Weiss, 1992;
Reynolds et al., 1992
). These
cells have the ability to self-renew in the presence of epidermal growth
factor (EGF) (Reynolds and Weiss,
1996
) and basic fibroblast growth factor
(Ciccolini and Svensden, 1998
),
forming spheric cell aggregates called neurospheres. Moreover, upon removal of
growth factors and adhesion onto a coated support, cells migrate out of the
sphere and form layers of differentiated neurons, astrocytes and
oligodendrocytes. Neurospheres can be regarded, therefore, as a simplified in
vitro model of CNS differentiation. Finally, neural progenitors expanded in
vitro can be grafted into the brain of recipient animals and have been shown
to differentiate in vivo (Svensden et al.,
1996
; Vescovi et al.,
1999
; Temple,
2001
). It is not known whether multipotent neural progenitor cells
need to be coupled and whether coupling territories are modulated during cell
differentiation. To address these issues, we have isolated neural progenitors
from the mouse embryonic striatal ventricular zone (SVZ) and found that
proliferating cells located in adherent neurospheres exhibited a strong
intercellular coupling. By contrast, only astrocytes were coupled among the
outgrowth of differentiating and migrating cells. In addition, we found that
the two cell populations that were coupled expressed Cx43, which became
predominantly phosphorylated after differentiation. Finally, treatment of
neurospheres with the gap junction inhibitor 18-ß-glycyrrhetinic acid
(ßGA), strongly reduced the viability of both proliferating and
differentiating neural cells. These observations demonstrate that junctional
coupling is widespread among CNS progenitors and substantiate the hypothesis
for an active role of Cx43 and intercellular communication during
proliferation and differentiation of neural progenitors.
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Materials and Methods |
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Preparation, growth and differentiation of neurospheres
Pregnant C57BL/6J mice at a gestational age of 14.5 to 15 days were killed
by cervical dislocation, and embryos were transferred in Petri dishes
containing ice-cooled HBSS (Hanks balanced solution without Ca2+
and Mg2+). Striata were dissected from the embryos and mechanically
dissociated as previously described
(Reynolds et al., 1992;
Ben-Hur et al., 1998
). The
viability of the single cell suspension was assessed using a 0.4% Trypan blue
solution (Gibco-BRL). Viable cells were seeded at a concentration ranging from
105 to 2.5x105 cells/ml in uncoated T75 tissue
culture flasks. Culture medium (hereafter referred to as standard medium)
consisted of DMEM-F12 supplemented with B27 (1/50 dilution) and contained
0.524 mg/ml stabilized glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin
and 20 ng/ml human recombinant EGF. Cells were cultured at 37°C in a
humidified atmosphere of 90%O2/10%CO2, and fresh EGF was
added every two days for the first 8 days, during which progenitors grew into
floating neurospheres. Subsequently, two thirds of the culture medium was
replaced twice a week. Neurospheres were passaged by centrifugation at 200
g for 5 minutes; the pellet was resuspended in one third of
the final volume, gently dissociated by pipetting and transferred into flasks
containing two thirds of the volume of fresh medium. Each neurosphere
preparation was kept in culture up to a maximum of one month, which
corresponded to four cell passages.
Differentiation of neural progenitors was induced by transferring floating neurospheres to an adhesive support and withdrawing EGF from standard medium. Briefly, glass coverslips, plastic flasks and Petri dishes were coated successively with poly-D-lysine (10 µg/ml in water) and fibronectin (10 µg/ml in PBS). An average of 10 floating neurospheres kept for a minimum of 10 days (one passage) and up to 3 weeks (three passages) in culture were transferred in 20 µl of standard culture medium onto freshly coated coverslips. For the large scale preparation of adherent neurospheres needed for biochemical analysis, floating neurospheres were collected by centrifugation (5 minutes at 200 g), resuspended in 2 ml culture medium and transferred onto coated plasticware. Neurospheres were incubated for 2 hours to initiate cell adhesion, and culture medium without EGF was gently added. Unless otherwise mentioned, adherent neurospheres were cultured for 8 days, during which medium was changed (half the volume) twice.
Dye coupling experiments
The incidence of cell-cell communication was assessed by microinjecting the
membrane-impermeant tracer Lucifer Yellow (LY; Mr 443)
into single cells located in different areas of neurospheres that had been
adherent for the specified times. Just before injections, neurospheres that
were adherent to coated glass coverslips were transferred onto the heated
(37°C) stage of an inverted microscope (Zeiss IM35), and 10 mM Hepes was
added to the culture medium. Electrodes were pulled to a resistance of 50-60
M and filled with a 4% solution of LY in 1 M LiCl. Following the
impalement of individual cells, the tracer was injected by iontophoresis,
applying square pulses of 0.1 nA amplitude, 900 msecond duration and 0.5 Hz
frequency for 5 minutes. Each injection site was photographed with Ektachrome
400 daylight film (Kodak, Rochester, NY) under phase contrast and
epifluorescence illumination, using appropriate filters, before and/or after
pulling out the electrode. Coupling was defined as LY diffusion from the
injected cell to at least one adjacent cell. Microinjected neurospheres were
fixed in 4% paraformaldehyde for 30 minutes at room temperature, rinsed three
times in phosphate-buffered saline (PBS) and stored at 4°C for subsequent
immunocytochemical analysis (see below).
Uncoupling experiments
Single cell suspensions (3x105 cells/ml) of striatal
progenitors in standard culture medium were aliquotted in 1.5 ml tubes
containing either the gap junction inhibitor ßGA
(Davidson et al., 1986) or its
inactive analog GZA at the specified final concentrations. Because of their
lipophilic nature, both drugs were dissolved in a mixture of dimethyl
sulfoxide (DMSO) and ethanol (3:2, vol:vol), pre-warmed at 37°C and mixed
by vortexing before addition to the medium (final concentrations of DMSO and
ethanol did not exceed 0.1%). Solvent alone was added to control cultures. 100
µl of cell suspension per well was distributed in 96-well plates (eight
wells/condition), and cell viability was measured three days later, in the
absence of any medium change. Reversibility was assessed on floating
neurospheres at the end of the 3 day incubation by adding two volumes of fresh
standard culture medium. Four days after dilution of the drugs, neurospheres
were examined with an inverted microscope and photographs were taken for each
condition. To assess cell viability on adherent cells, floating neurospheres
expanded in culture for 10 days were spun at 200 g for 5
minutes, dissociated as a single cell suspension and eventually aliquoted
(1x105 cells/ml) into 96-well plates that had been previously
coated with poly-D-lysine and fibronectin. For long-term treatment of adherent
neurospheres, 0.25% serum albumin (BSA) was added to the culture medium to
avoid the toxic effects of DMSO. Adherent neurospheres were cultured for eight
days, during which ßGA (50 µM) and DMSO were added every two days, and
half the medium was replaced with fresh medium every three days. To test
whether the uncoupling agent had an effect on differentiated cells,
neurospheres were first cultured for 3 days under control conditions, to allow
differentiation of neural progenitor cells, before adding ßGA (50 µM)
for the next 5 days, as described above.
Cell proliferation and viability assays
Adherent neurospheres were incubated for 24 hours in the presence of 10
µM BrdU, fixed for 30 minutes at room temperature in ethanol/acetic acid
(95%/5%, vol/vol) and then processed for immunostaining with a mouse IgG1
antibody (1/50; Becton Dickinson, San Jose, CA), as described elsewhere
(Ben-Hur et al., 1998). Viable
cells in either floating or adherent neurospheres were quantified
colorimetrically by the MTT assay, in which the tetrazolium salt is cleaved in
the mitochondria of metabolically active cells to form a precipitable formazan
dye (Mosmann, 1983
). MTT was
added at a final concentration of 1 µg/ml to each well for 4 hours at
37°C, and precipitates were solubilized in MTT dissolving buffer (100
µl/well). Plates were first incubated 5 minutes at 37°C, followed by a
10 minute period at room temperature on a shaking stage to allow color
development. Differences in optical densities between 570 nm and 630 nm were
measured spectrophotometrically.
Antibodies
Differentiating oligodendrocytes were detected with the O4 mouse monoclonal
antibody (IgM 1:20; a kind gift from Susan Barnett, Glasgow, Scotland).
Neurons were identified with TujI, a mouse IgG2a antibody raised against the
neuron specific ß3-tubulin (1:500; Babco, Richmond, CA). Astrocytes were
labeled with either a rabbit polyclonal (1/200; DAKO, Glostrup, Denmark) or a
mouse monoclonal IgG1 (1:100; Chemicon, Temecula, CA) antibody raised against
glial fibrillary acidic protein (GFAP). A mouse monoclonal IgG1 and a rabbit
polyclonal IgG against Cx43 (both from Zymed, San Francisco, CA) were employed
for immunocytochemistry (at a dilution of 1:250 and 1:500, respectively) and
western blotting (at a dilution of 1:3,000). Fluorescein-conjugated goat
anti-mouse IgG and anti-rabbit IgG, rhodamine-conjugated goat anti-mouse IgG,
biotin-conjugated anti-rabbit IgG and
streptavidin-7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated donkey
anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West
Grove, PA).
Immunocytochemistry
Floating neurospheres were fixed 4 hours at 4°C in 2% paraformaldehyde
in PBS, washed in PBS, incubated overnight at 4°C in a 15% sucrose
solution and eventually embedded in a drop of Tissue-Tek compound (Miles
Scientific, Naperville, IL). Specimens were immediately frozen on dry ice and
stored at -20°C until frozen sections, cut at the 10 µm setting of a
CM-3000 cryostat (Leica, Nussloch, Germany), were collected on coated slides
and processed for immunocytochemistry. Adherent cells were fixed in 4%
paraformaldehyde, rinsed repeatedly in PBS and incubated with primary and
secondary antibodies as indicated below. For Cx43 labeling, cells were fixed
in 2% paraformaldehyde, then washed and permeabilized with 0.1% Triton X-100
in blocking solution for 30 minutes.
All samples were incubated overnight at 4°C in a humidified chamber
with primary antibodies diluted in blocking solution (PBS supplemented with 5%
normal goat serum and 5% normal donkey serum), rinsed repeatedly in PBS and
then stained for 30 minutes at room temperature with the cognate secondary
antibodies diluted in blocking solution. Triple immunolabeling was carried out
using primary mouse antibodies of the IgM and IgG classes and a rabbit
antibody, as previously described
(McKinnon et al., 1990;
Ben-Hur et al., 1998
). After
several washes in PBS, immunostained cells were preserved in Vectashield
mounting medium (Vector Laboratories, Inc., Burlingame, CA). The specificity
of the immunofluorescent staining was assessed for each experimental condition
by performing the first incubation in the absence of primary antibodies. No
staining was observed under such conditions.
Photographs were taken with a Leica microcope (DMRB model) equipped with a Coolsnap camera system (Princeton Instruments, Evry, France), stored on a PC and processed with Photoshop 5.5 software (Adobe Systems, San Jose, CA). Confocal microscopy was performed using an Axiovert LSM 510 microscope (Zeiss, Jena, Germany) equipped with 63x 1.4 NA and 100x 1.4 NA objective lenses and three separate laser beams. Samples were labeled with fluorescein (FITC)- and/or rhodamine (TRITC)-conjugated secondary antibodies. Double-labeled samples were analyzed either simultaneously or sequentially. In either case, FITC was excited by the blue beam and detected through an interferential narrow band filter (BP 505-550 nm), whereas TRITC was excited by the red beam and detected through a long pass filter (LP 650 nm).
Western blotting and dephosphorylation assay
Protein extracts were prepared in a lysis buffer containing 50 mM NaCl, 50
mM Tris-HCl (pH 8), 0.1% SDS, 1% NP-40, 0.5% deoxycholate, supplemented with
10 µg each chymostatin, leupeptin and pepstatin and 10 KU/ml Trasylol.
Neurospheres were solubilized in 2 ml lysis buffer (for either a pellet of
floating neurospheres or neurospheres adherent to a T75 flask) by repeated
aspiration (20 times) through a 23-gauge needle. Crude extracts were
centrifuged at 4°C for 30 minutes at 10,000 g, and
supernatants were then submitted to protein quantification using the Micro BCA
protein assay kit, according to the manufacturer's recommendations. Equal
amounts of total protein were incubated overnight at 37°C in the presence
of 100 units of calf intestinal alkaline phosphatase. Control reactions were
carried out with phosphatase buffer only or in the presence of phosphatase
inhibitors (2 mg/ml Na-orthovanadate, 10 mM EDTA and 10 mM PO4), as
previously described (Musil et al.,
1990a). Samples were quenched by addition of gel loading buffer
(0.025 M Tris pH 6.8, 0.5% SDS, 1% ß-mercaptoethanol, 0.025% bromophenol
blue, 17.5% glycerol) and aliquots were loaded on a 12.5% polyacrylamide
SDS-gel. After separation, proteins were transferred to nitrocellulose
membranes (Protran; Schleicher & Schuell, Keene, NH). Membranes were
saturated for 30 minutes in PBS containing 5% non-fat dried milk and 0.1%
Tween and subsequently incubated overnight with either the monoclonal or
polyclonal anti-Cx43 antibody (Zymed) diluted 1:3,000 in saturation buffer.
After extensive washes in PBS containing 0.1% Tween, membranes were probed for
1 hour with the corresponding horseradish-peroxidase-conjugated secondary
antibodies (Chemicon) diluted in saturation buffer (anti-rabbit: 1:10,000;
anti-mouse: 1:5,000), rinsed again in PBS-Tween and revealed with the
SuperSignal West chemiluminescent substrate kit, according to the
manufacturer's instructions. The time of incubation in ECL detection reagents
and exposure (typically 1 minute) to Hyperfilm (Amersham-Pharmacia,
Buckinghamshire, UK) were identical for all experimental conditions.
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Results |
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Cell proliferation in neurospheres
To determine whether neurospheres maintained the potential to proliferate
following removal of growth factors and adhesion, adherent neurospheres were
incubated for 24 hours in the presence of 10 µM BrdU and then processed for
immunostaining. After 7 days of adhesion, proliferating cells were
concentrated in the neurosphere, whereas only scattered BrdU-positive cells
were observed in the outgrowth formed by migrating cells
(Fig. 4). Similar observations
were made when spheres had been adherent for only 45 minutes and were composed
of neural progenitor cells expressing nestin, an intermediate filament protein
of neuroepithelial stem cells (data not shown). Thus, cells within the borders
of spheres consisted of a mixed population of undifferentiated neural
progenitors and neural precursor cells committed to a specific cell type of
which they do not yet express the morphological and immunocytochemical
markers. For the sake of clarity, we hereafter refer to these two populations
of proliferating cells as neural progenitor cells. By contrast, cell layers
are composed of differentiated, mainly non-proliferating cells.
|
Effect of 18-ß-glycyrrhetinic acid (ßGA) on neural
progenitor cells
To assess whether junctional coupling was involved in the growth of neural
progenitors, floating neurospheres were incubated in the presence of
increasing concentrations of ßGA. The size of neurospheres was
drastically reduced by ßGA (Fig.
5C,D), in comparison with cultures exposed to equal concentrations
of the inactive analog glycyrrhyzic acid (GZA)
(Fig. 5A,B), solvent
(DMSO/ethanol) alone or mock-treated in standard medium (data not shown). To
verify the specificity of the ßGA effect, we tested its reversibility by
incubating floating neurospheres for an additional 4 day period in standard
culture medium without any drugs. At the end of this period, neurospheres
pre-treated with 25 µM and 50 µM ßGA
(Fig. 5G,H) recovered to a size
similar to that of spheres incubated with either GZA
(Fig. 5E,F) or solvent (data
not shown), suggesting that progenitors temporarily exposed to ßGA could
still proliferate as untreated cells. By contrast, cells incubated in the
presence of 100 µM ßGA, but not with the same concentration of GZA,
were unable to grow and form neurospheres even after removal of the uncoupling
drug (data not shown). Thus, in all other experiments, ßGA was added at
the maximally effective concentration of 50 µM.
|
To assess whether junctional coupling was involved in the differentiation
of neural progenitors, adherent neurospheres were incubated for 8 days in the
presence of 50 µM ßGA. Under these conditions, both cell density and
morphological features of migrating cells were greatly perturbed
(Fig. 6E-H) when compared with
DMSO-treated cultures (Fig.
6A-D), which were indistinguishable from mock-treated cells (data
not shown). Although a majority of cells were still able to acquire antigenic
determinants of either the astrocytic, neuronal or oligodendrocytic phenotype,
they also exhibited strikingly different features compared with mock- and
DMSO-treated cells. Thus, GFAP-positive cells displayed thin, elongated
processes (compare Fig. 6B with
F), whereas oligodendrocytes were less branched (compare
Fig. 6D with G), and
ß3-tubulin-positive neurons appeared as round cells with few processes
(compare Fig. 6D with 6H).
Furthermore, treatment of neurospheres with 50 µM carbenoxolone, another
uncoupling agent (Rozental et al.,
2001), induced modifications of both cell density and morphology
that were comparable to those observed with ßGA (data not shown). By
contrast, these alterations were not observed when neurospheres were allowed
to differentiate before being exposed to the same concentration (50 µM) of
either ßGA (Fig. 6I-L) or
carbenoxolone (data not shown). Taken together, our results suggest that the
effects on the growth and differentiation of neural progenitor cells are
specifically due to disruption of junctional coupling.
|
Because ßGA reduced cell numbers in both floating and adherent neurospheres, we quantified cell viability following a 3 day incubation in the presence of certain drugs (Fig. 7). ßGA caused a dose-dependent and statistically significant decrease in the number of viable cells, in both floating and adherent neurospheres (Fig. 7). By contrast, neither the solvent DMSO/ethanol nor the inactive analog GZA modified cell viability with respect to that of mock-treated cultures (Fig. 7).
|
To assess the effect of ßGA on coupling, astrocytes grown out of 21-day-old adherent neurospheres, which had been incubated for 6 hours in the presence or absence of 50 µM ßGA, were microinjected with LY. As compared to controls, astrocytes treated with ßGA were much less coupled (data not shown).
Cx43 expression in neural progenitor cells and differentiating
astrocytes
We started our investigation of the molecular identity of connexins in
neural progenitors by focusing on Cx43, which is the main component of
astrocytic gap junctions. Floating neurospheres grown for 10 days in the
presence of EGF were immunolabeled with a monoclonal antibody directed against
Cx43. Examination of sections by confocal microscopy revealed an intense Cx43
labeling of the vast majority of undifferentiated cells
(Fig. 8B). To investigate the
expression of Cx43 among differentiating cells, neurospheres were allowed to
adhere for 8 days and then double-labeled with antibodies against Cx43 and
cell-specific markers. We found that all cells labeled with Cx43 antibodies
were also positive for GFAP (Fig.
8C), whereas we never observed a colocalization of Cx43 with
either the O4 antigen of oligodendrocytes or ß3-tubulin-positive neurons
of the cell layers (data not shown).
|
We next analyzed the time course of Cx43 and GFAP expression in differentiating neurospheres (Fig. 9). Shortly (30 minutes) after adhesion, virtually each cell in the neurosphere expressed Cx43, whereas only a few GFAP-positive processes were detectable (Fig. 9A,B). Three hours after adhesion, Cx43 expression was mainly concentrated within the sphere of the cells, but was also detected in a few cells of the outgrowing layers. At the same time point, Cx43-and GFAP-positive cells were seen at the periphery of the sphere, where the migrating cells were beginning to form the outgrowing layers (Fig. 9C,D). Thus, at early stages of in vitro differentiation, Cx43 was expressed in both proliferating cells and in cells committed to an astrocytic fate. After longer adhesion times (8 days), Cx43 was still detectable within the sphere, as well as in differentiated astrocytes of the migrating cell layers (Fig. 9E,F).
|
Phosphorylation of Cx43 in adherent neurospheres
The expression pattern of Cx43 suggests that Cx43 is regulated during
differentiation of neural progenitor cells. Because Cx43 is
post-translationally phosphorylated, we analyzed whether changes in the
phosphorylation state of Cx43 occurred during differentiation
(Fig. 10). Equal amounts of
protein extracted from floating and adherent neurospheres were studied by
western blotting using two anti-Cx43 antibodies: a rabbit polyclonal,
recognizing both the phosphorylated and nonphosphorylated forms of Cx43, and a
mouse monoclonal specific for the latter form of the protein
(Nagy et al., 1997). Cell
lysates were separated by SDS-gel electrophoresis after no treatment, an
incubation with alkaline phosphatase or an incubation with this enzyme in the
presence of an excess of phosphatase inhibitors. In protein extracts from
floating neurospheres (10 days), both antibodies reacted with a band of
similar electrophoretic mobility, which was not perturbed by the alkaline
phosphatase treatment (Fig.
10, lanes 1-3). In proteins from adherent neurospheres, the
polyclonal antibody detected a broader band, which exhibited a slower
electrophoretic mobility (Fig.
10, lane 4). Phosphatase treatment shifted Cx43 mobility towards
faster migrating forms, an effect that was reduced in the presence of
phosphatase inhibitors (Fig.
10, lanes 5-6). Under these experimental conditions, the
monoclonal antibody, which recognizes nonphosphorylated Cx43, did not detect
Cx43 in adherent neurospheres, unless protein extracts had been pretreated
with alkaline phosphatase (Fig.
10, compare lanes 4 with 5 in panel B). Consistent with these
findings, immunolabeling performed on adherent neurospheres (8 days old) with
the polyclonal anti-Cx43 antibody revealed a strong signal at the membrane of
both cells located within the sphere, as well as differentiated astrocytes in
the outgrowth layers. By contrast, the same two cell populations were weakly
labeled when the monoclonal antibody against Cx43 was used. These findings
suggest that Cx43 is mainly expressed as a nonphosphorylated form by growing
neural progenitors, whereas the phosphorylated species predominates following
in vitro differentiation.
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Discussion |
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Cell coupling between neural progenitors
Microinjection of LY demonstrated that cells located within the neurosphere
were always strongly coupled. Although injections were performed on
neurospheres that had been adhering for various periods of time, virtually all
these cells were nestin positive up to 2 hours after adhesion, confirming that
they were undifferentiated progenitor cells
(Lendahl et al., 1990). As
adhesion time increased, there was a decrease in the number of cells in the
neurosphere and a parallel expansion of the outgrowth composed of migrating
cells, resulting in an increased proportion of differentiated cells. The
population of undifferentiated cells, which was an area of active
proliferation compared with the outgrowth layers, remained coupled throughout
the adhesion period. To our knowledge, there has been no previous report
describing that gap junction communication between cells retains the ability
to differentiate into the three main CNS cell types. These findings suggest
that cell-cell coupling may be relevant to the maintenance of cells in a
proliferative state, which may require synchronization in the center of the
sphere. This possibility is in agreement with the observation that neural
precursors from the ventricular zone couple and uncouple in a dynamic fashion
throughout the cell cycle (Bittman and
LoTurco, 1999
).
Cell coupling between differentiated cells
As soon as migrating cells acquired an astrocytic morphology and GFAP
expression, homocellular coupling was invariably detected. This observation
confirms studies performed in cultures and brain slices that have demonstrated
that astrocytes establish a functional syncytium through gap junction channels
(Giaume and Venance, 1998;
Dermietzel and Spray, 1998
).
By contrast, oligodendrocytes, and neurons did not allow transfer of LY to
adjacent cells, in spite of the fact that microinjected cells were in apparent
contact with many neighbors. Together, these findings suggest that
proliferating cells and differentiated astrocytes form two distinct
communication compartments, from which oligodendrocytes and neurons appear to
be excluded. Although the occurrence of heterocellular coupling between
neurons and astrocytes has been reported
(Nedergaard, 1994
;
Fróes and Campos de Carvalho,
1998
; Fróes et al.,
1999
; Alvarez-Maubecin et al.,
2000
), no morphological evidence of gap junctions between these
two cell types has yet been found (Rash et
al., 2001
). It is possible that the discrepancy between the
functional and the morphological observations reflects the limits of the
techniques or, alternatively, that heterocellular coupling occurs during a
narrow temporal window (Fróes et
al., 1999
) and/or in restricted brain areas
(Alvarez-Maubecin et al.,
2000
).
Neuronal coupling via gap junctions is well established both in vitro and
in vivo (LoTurco and Kriegstein,
1991; Peinado et al.,
1993
; Donahue et al.,
1998
; Rozental et al.,
1998
; Roerig and Feller,
2000
; Venance et al.,
2000
). In our system, however, neurons expressing the post-mitotic
marker ß3-tubulin were not coupled among themselves nor to glia, as
judged by LY exchange. One explanation is that coupling is transient during
cell migration and, therefore, more difficult to detect in cell cultures than
in brain slices. Moreover, we cannot exclude the possibility that tracer
molecules other than LY may be more permeable to neuronal connexins. However,
intercellular communication has been detected between differentiating neurons
using LY (LoTurco and Kriegstein,
1991
), and in a variety of systems this tracer is as sensitive as
neurobiotin to dye coupling (Pastor et
al., 1998
; Meda,
2000
). Our observations are also supported by those made in
co-cultures of rat striatal astrocytes and neurons, where coupling was
restricted to astrocytes, as demonstrated by both LY microinjection and patch
clamp techniques (Rouach et al.,
2000
).
The presence of coupling between oligodendrocytes is still debated. Thus,
oligodendrocytes of grey, but not white, matter exhibited homocellular
coupling (Pastor et al.,
1998), as did fully differentiated cells in culture
(Venance et al., 1995
;
Dermietzel et al., 1997
). Our
results support the notion that, during differentiation, oligodendrocytes are
not engaged in direct cell-cell communication. Although it has been clearly
established that oligodendrocytes express more than one connexin type
(Dermietzel et al., 1989
;
Scherer et al., 1995
;
Kunzelmann et al., 1997
), it
is possible that these connexins only form reflexive gap junctions that
provide a short-cut pathway for the transfer of signals between different
compartments within the same cell (Scherer
et al., 1995
).
Cell uncoupling and viability
A pharmacological blockade of intercellular communication by ßGA
produced a striking, dose-dependent inhibition of the viability of precursor
cells, as well as a change in cell morphology. Several lines of evidence agree
with a specific effect for ßGA as the result of its uncoupling
properties. First, ßGA effects were not mimicked by either the inactive
analog GZA or the solvent used to deliver the drug; second, morphological
alterations of differentiated cells were similar in the presence of another
uncoupler, carbenoxolone. More importantly, removal of 50 µM ßGA led
floating neurospheres to recover to a size similar to that of the controls. It
is intriguing that treatment of adherent neurospheres with ßGA affected
cell density and the morphology of the uncoupled differentiating cells:
neurons and oligodendrocytes. A possible explanation is that the absence of a
well organized astrocytic layer has a profound impact on the survival and
differentiation of the other cell types
(Hooghe-Peters et al., 1981),
which, being uncoupled, should have not been directly affected by bGA. Since
neither ßGA nor carbenoxolone perturbed the morphology and viability of
cells that had been allowed to differentiate (namely, when the drugs were
applied after 3 days of adhesion), an alternative hypothesis may take into
account the effects observed after continuous treatment with ßGA.
Uncoupling during the early steps of differentiation may affect the viability,
as well as the proper morphological differentiation of surviving progenitors
and precursors, thereby leading to alterations in morphology and survival of
their progeny. In the presence of ßGA, the strong reduction in the size
of floating neurospheres and in the density of the layers formed by migrating
cells could be accounted for by either a decreased rate of proliferation
and/or an increased cell death. Our data cannot distinguish between these two
mechanisms but suggest that cell communication is of paramount importance to
orchestrate the life cycle of neural progenitors and promote their viability,
as suggested in the case of early migratory neural crest cells
(Bannerman et al., 2000
).
Further investigations are needed to specifically address this issue.
Cx43 in neurospheres: expression and post-translational
modification
Cx43 protein was expressed by most cells of floating neurospheres and was
still present in neural progenitor and precursor cells, even after long
adhesion periods under culture conditions promoting differentiation. These
results indicate that Cx43 plays a major role in connexin-dependent
communication of neural progenitors.
Following migration and differentiation, Cx43 was confined to astrocytes. This segregation prompted us to compare the time course of Cx43 and GFAP expression following adhesion. GFAP was initially detected in the few cells that accumulated at the periphery of neurospheres, and Cx43 was found in these GFAP-positive cells migrating out of the sphere. At later time points, neurospheres contained Cx43-positive and GFAP-negative cells, whereas in the outgrowth, Cx43 was exclusively expressed by GFAP-positive astrocytes. The two areas of Cx43 expression were also those exhibiting dye coupling, suggesting that Cx43 mediates cell communication in these compartments and also contributes to isolating them from cells committed to either a neuronal or an oligodendrocytic fate.
Biochemical analysis demonstrated that a non-phosphorylated form of Cx43
was prevalent in neural progenitors, whereas, following differentiation, there
was a massive shift towards phosphorylated species. This was demonstrated by
comparing the ability of two anti-Cx43 antibodies, a mouse monoclonal specific
for the non-phosphorylated form (Nagy et
al., 1997) and a rabbit polyclonal also recognizing the
phosphorylated species, to detect Cx43 by western blotting in floating and
adherent neurospheres. The differences observed, namely the lack of signal
from adherent neurospheres with the monoclonal antibody, unless protein
extracts had been pre-treated with alkaline phosphatase, were further
corroborated by immunocytochemical analysis of adherent neurospheres. Under
these conditions, both antibodies recognized the same cells located within the
sphere and in the outgrowth, although a much stronger staining was present
with the polyclonal antibody. Phosphorylation of Cx43 has been implicated in
several aspects of channel function (Musil
et al., 1990b
; Moreno et al.,
1992
; Kwak et al.,
1995
; Postma et al.,
1998
; Warn-Cramer et al.,
1998
) and could be tightly linked to the program of cell
differentiation.
In summary, coupling among neural progenitors and the reduced viability of
both proliferating and differentiating neural cells observed during
pharmacological uncoupling, which presumably affects the channels made by all
connexins, supports a central role for intercellular communication during CNS
development. In this context, it is puzzling that no major CNS abnormality has
been detected in Cx43 knockout mice
(Reaume et al., 1995). Other
connexins most probably help to define communication compartments during the
early steps of differentiation of neural progenitors and provide a
compensatory mechanism in the case of Cx43 loss. Our preliminary data show
that at least eight other connexins are detectable in neurospheres (N.D.,
D.G., V.C. et al., unpublished), offering a unique experimental model in which
this compensatory possibility can be tested.
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Alvarez-Maubecin, V., Garcia-Hernandez, F., Williams, J. T. and
van Bockstaele, E. J. (2000). Functional coupling between
neurons and glia. J. Neurosci.
20,4091
-4098.
Bani-Yaghoub, M., Bechberger, J. F., Underhill, T. M. and Naus, C. C. (1999a). The effects of gap junction blockage on neuronal differentiation of human NTera2/clone D1 cells. Exp. Neurol. 156,16 -32.[Medline]
Bani-Yaghoub, M., Underhill, T. M. and Naus, C. C. (1999b). Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells. Dev. Genet. 24,69 -81.[Medline]
Bannerman, P., Nichols, W., Puhalla, S., Oliver, T., Berman, M. and Pleasure, D. (2000). Early migratory rat neural crest cells express functional gap junctions: evidence that neural crest cell survival requires gap junction function. J. Neurosci. Res. 61,605 -615.[Medline]
Ben-Hur, T., Rogister, B., Murray, K., Rougon, G. and
Dubois-Dalcq, M. (1998). Growth and fate of PSA-NCAM+
precursors of the postnatal brain. J. Neurosci.
18,5777
-5788.
Bittman, K. S. and LoTurco, J. J. (1999).
Differential regulation of connexin 26 and 43 in murine neocortical
precursors. Cereb. Cortex
9, 188-195.
Bittman, K., Owens, D. F., Kriegstein, A. R. and LoTurco, J.
J. (1997). Cell coupling and uncoupling in the ventricular
zone of developing neocortex. J. Neurosci.
17,7037
-7044.
Bruzzone, R., White, T. W. and Paul, D. L. (1996). Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1-27.[Abstract]
Ciccolini, F. and Svendsen, C. N. (1998).
Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth
factor (EGF) responsiveness in mouse striatal precursor cells: identification
of neural precursors responding to both EGF and FGF-2. J.
Neurosci. 18,7869
-7880.
Davidson, J. S., Baumgarten, I. M. and Harley, E. H. (1986). Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem. Biophys. Res. Commun. 134,29 -36.[Medline]
Dermietzel, R. and Spray, D. C. (1998). From neuro-glue (`Nervenkitt') to glia: a prologue. Glia 24, 1-7.[Medline]
Dermietzel, R., Traub, O., Hwang, T. K., Beyer, E., Bennett, M. V. L., Spray, D. C. and Willecke, K. (1989). Differential expression of three gap junction proteins in developing and mature brain tissues. Proc. Natl. Acad. Sci. USA 86,10148 -10152.[Abstract]
Dermietzel, R., Farooq, M., Kessler, J. A., Althaus, H., Hertzberg, E. L. and Spray, D. C. (1997). Oligodendrocytes express gap junction proteins connexin32 and connexin45. Glia 20,101 -114.[Medline]
Donahue, L. M., Webster, D. R., Martinez, I. and Spray, D. C. (1998). Decreased gap-junctional communication associated with segregation of the neuronal phenotype in the RT4 cell-line family. Cell Tissue Res. 292,27 -35.[Medline]
Fróes, M. M. and Campos de Carvalho, A. C. (1998). Gap junction-mediated loops of neuronal-glial interactions. Glia 24,97 -107.[Medline]
Fróes, M. M., Correia, A. H., Garcia-Abreu, J., Spray, D.
C., Campos de Carvalho, A. C. and Neto, V. M. (1999).
Gap-junctional coupling between neurons and astrocytes in primary central
nervous system cultures. Proc. Natl. Acad. Sci. USA
96,7541
-7546.
Giaume, C. and Venance, L. (1998). Intercellular calcium signaling and gap junctional communication in astrocytes. Glia 24,50 -64.[Medline]
Hooghe-Peters, E. L., Meda, P. and Orci, L. (1981). Co-cultures of nerve cells and pancreatic cells. Dev. Brain Res. 1,287 -292.
Kandler, K. and Katz, L. C. (1995). Neuronal coupling and uncoupling in the developing nervous system. Curr. Opin. Neurobiol. 5,98 -105.[Medline]
Kumar, N. M. and Gilula, N. B. (1996). The gap junction communication channel. Cell 84,381 -388.[Medline]
Kunzelmann, P., Blumcke, I., Traub, O., Dermietzel, R. and Willecke, K. (1997). Coexpression of connexin45 and -32 in oligodendrocytes of rat brain. J. Neurocytol. 26, 17-22.[Medline]
Kwak, B. R., Hermans, M. M., de Jonge, H. R., Lohmann, S. M., Jongsma, H. J. and Chanson, M. (1995). Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. Mol. Biol. Cell 6,1707 -1719.[Abstract]
Lendahl, U., Zimmermann, L. B. and McKay, R. D. G. (1990) CNS stem cells express a new class of intermediate neurofilament protein. Cell 60,585 -595.[Medline]
Lo, C. W. (1996). The role of gap junction membrane channels in development. J. Bioenerg. Biomembr. 28,379 -385.[Medline]
LoTurco, J. J. and Kriegstein, A. R. (1991). Clusters of coupled neuroblasts in embryonic cortex. Science 252,563 -566.[Medline]
McKinnon, R. D., Matsui, T., Dubois-Dalcq, M. and Aaronson, S. A. (1990). FGF modulates the PDGF-driven pathway of oligodendrocyte development. Neuron 5, 603-614.[Medline]
Meda, P. (2000). Probing the function of connexin channels in primary tissues. Methods 20,232 -244.[Medline]
Menezes, J. R., Froés, M. M., Moura Neto, V. and Lent, R. (2000). Gap junction-mediated coupling in the postnatal anterior subventricular zone. Dev. Neurosci. 22, 34-43.[Medline]
Miragall, F., Albiez, P., Bartels, H., de Vries, U. and Dermietzel, R. (1997). Expression of the gap junction protein connexin43 in the subependymal layer and the rostral migratory stream of the mouse: evidence for an inverse correlation between intensity of connexin43 expression and cell proliferation activity. Cell Tissue Res. 287,243 -253.[Medline]
Moreno, A. P., Fishman, G. I. and Spray, D. C. (1992). Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys. J. 62,51 -53.[Medline]
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.[Medline]
Musil, L. S., Beyer, E. C. and Goodenough, D. A. (1990a). Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membr. Biol. 116,163 -175.[Medline]
Musil, L. S., Cunningham, B. A., Edelman, G. M. and Goodenough, D. A. (1990b). Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell Biol. 111,2077 -2088.[Abstract]
Nadarajah, B., Jones, A. M., Evans, W. H. and Parnavelas, J.
G. (1997). Differential expression of connexins during
neocortical development and neuronal circuit formation. J.
Neurosci. 17,3096
-3111.
Nagy, J. I., Li, W. E., Roy, C., Doble, B. W., Gilchrist, J. S., Kardami, E. and Hertzberg, E. L. (1997). Selective monoclonal antibody recognition and cellular localization of an unphpsphorylated form of Cx43. Exp. Cell Res. 236,127 -136.[Medline]
Naus, C. C. and Bani-Yaghoub, M. (1998). Gap junctional communication in the developing central nervous system. Cell Biol. Int. 22,751 -763.[Medline]
Nedergaard, M. (1994). Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263,1768 -1771.[Medline]
Pastor, A., Kremer, M., Moller, T., Kettenmann, H. and Dermietzel, R. (1998). Dye coupling between spinal cord oligodendrocytes: differences in coupling efficiency between gray and white matter. Glia 24,108 -120.[Medline]
Peinado, A., Yuste, R. and Katz, L. C. (1993). Extensive dye coupling between rat neocortical neurons during the period of circuit formation. Neuron 10,103 -114.[Medline]
Postma, F. R., Hengeveld, T., Alblas, J., Giepmans, B. N.,
Zondag, G. C., Jalink, K. and Moolenaar, W. H. (1998). Acute
loss of cell-cell communication caused by G protein-coupled receptors: a
critical role for c-Src. J. Cell Biol.
140,1199
-1209.
Rash, J. E., Yasumura, T., Dudek, F. E. and Nagy, J. I.
(2001). Cell-specific expression of connexins and evidence of
restricted gap junctional coupling between glial cells and between neurons.
J. Neurosci. 21,1983
-2000.
Reaume, A. G., de Sousa, P. A., Kulkarni, S., Langille, B. L., Zhu, D., Davies, T. C., Juneja, S. C., Kidder, G. M. and Rossant, J. (1995). Cardiac malformation in neonatal mice lacking connexin43. Science 267,1831 -1834.[Medline]
Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255,1707 -1710.[Medline]
Reynolds, B. A. and Weiss, S. (1996). Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175, 1-13.[Medline]
Reynolds, B. A., Tetzlaff, W. and Weiss, S. (1992). A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J. Neurosci. 12,4565 -4574.[Abstract]
Roerig, B. and Feller, M. B. (2000). Neurotransmitters and gap junctions in developing neural circuits. Brain Res. Rev. 32,86 -114.[Medline]
Rouach, N., Glowinski, J. and Giaume, C.
(2000). Activity-dependent neuronal control of gap-junctional
communication in astrocytes. J. Cell Biol.
149,1513
-1526.
Rozental, R., Morales, M., Mehler, M. F., Urban, M., Kremer, M.,
Dermietzel, R., Kessler, J. A. and Spray, D. C. (1998).
Changes in the properties of gap junctions during neuronal differentiation of
hippocampal progenitor cells. J. Neurosci.
18,1753
-1762.
Rozental, R., Srinivas, M. and Spray, D. C. (2001). How to close a gap junction channel. Methods Mol. Biol. 154,447 -476.[Medline]
Scherer, S. S., Deschênes, S. M., Xu, Y. T., Grinspan, J. B., Fischbeck, K. H. and Paul, D. L. (1995). Connexin32 is a myelin-related protein in the PNS and CNS. J. Neurosci. 15,8281 -8294.[Abstract]
Svendsen, C. N., Clarke, D. J., Rosser, A. E. and Dunnett, S. B. (1996). Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. Exp. Neurol. 137,376 -388.[Medline]
Temple, S. (2001). Stem cell plasticitybuilding the brain of our dreams. Nat. Rev. Neurosci. 2,513 -520.[Medline]
Venance, L., Cordier, J., Monge, M., Zalc, B., Glowinski, J. and Giaume, C. (1995). Homotypic and heterotypic coupling mediated by gap junctions during glial cell differentiation in vitro. Eur. J. Neurosci. 7,451 -461.[Medline]
Venance, L., Rozov, A., Blatow, M., Burnashev, N., Feldmeyer, D.
and Monyer, H. (2000). Connexin expression in electrically
coupled postnatal rat brain neurons. Proc. Natl. Acad. Sci.
USA 97,10260
-10265.
Vescovi, A. L., Parati, E. A., Gritti, A., Poulin, P., Ferrario, M., Wanke, E., Frolichsthal-Schoeller, P., Cova, L., Arcellana-Panlilio, M., Colombo, A. and Galli, R. (1999). Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp. Neurol. 156,71 -83.[Medline]
Warn-Cramer, B. J., Cottrell, G. T., Burt, J. M. and Lau, A.
F. (1998). Regulation of connexin-43 gap junctional
intercellular communication by mitogen-activated protein kinase. J.
Biol. Chem. 273,9188
-9196.