1 Max-Planck-Institute of Neurobiology, Neuronal Specification, Am Klopferspitz
18a, D-82152 Martinsried, Germany
2 Institute of Anatomy, University of Freiburg, Albertstr.17, D-79104 Freiburg,
Germany
3 Department of Molecular Genetics, UT Southwestern, 5323 Harry Hines Blvd,
Dallas, TX 75390-9046, USA
4 University of Liège, Center for Cellular and Molecular Neurobiology, 17
Place Delcour, B-4020 Liège, Belgium
5 Instituto de Neurociencias UMH/CSIC, Campus de San Juan s/n, E-03550 San Juan
de Alicante, Spain
* Author for correspondence (e-mail: mgoetz{at}neuro.mpg.de)
Accepted 9 June 2003
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SUMMARY |
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Key words: Neurogenesis, reeler mutant, Dab1, Apoer2, Vldlr, Precursor morphology
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Introduction |
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We have previously identified the transcription factor Pax6 as a necessary
and sufficient determinant for the neurogenic lineage of cortical radial glia
(Götz et al., 1998;
Heins et al., 2002
). In
correlation to an almost complete loss of the neurogenic radial glia, Glast is
strongly reduced and the morphology of radial glia is affected in the
Pax6-mutant cortex (Götz et al.,
1998
; Heins et al.,
2002
). However, Pax6-deficient radial glia are still attached to
the pial surface, they still contain the brain lipid-binding protein (Blbp)
and neuronal migration is only affected at late developmental stages
(Caric et al., 1997
;
Götz et al., 1998
) (N.
Haubst and M.G., unpublished). Blbp had been suggested to promote the bipolar
morphology of radial glia when neurons attach in vitro
(Feng et al., 1994
;
Kurtz et al., 1994
;
Anton et al., 1997
). ErbB
receptors should be involved in mediating this neuron-glia signaling, but the
respective mouse mutants show only a minor phenotype in branching of radial
glia endfeet and still possess long radial glia processes
(Anton et al., 1997
). Thus, the
proposed role of Blbp in influencing the morphology of radial glia has so far
not been substantiated in vivo and the signals that regulate radial glia
process extension remain elusive.
We examined reelin as a candidate molecule for influences on radial glia
that are in close contact with reelin-secreting cells in the marginal zone
(MZ). Reelin is a large secreted glycoprotein, the absence of which has
profound effects on neuronal migration in the cerebral cortex and cerebellum,
but not the basal ganglia (D'Arcangelo et
al., 1995) (for reviews, see
Caviness et al., 1988
;
Curran and D'Arcangelo, 1998
;
Lambert de Rouvroit and Goffinet,
1998
). Reelin binds to the lipoprotein receptors apolipoprotein
receptor 2 (Apoer2; Lrp8- Mouse Genome Informatics) and the very low-density
lipoprotein receptor (Vldlr) (D'Arcangelo
et al., 1999
; Hiesberger et
al., 1999
), and their role as receptors for reelin has been
confirmed in vivo by the identical phenotype of mice lacking both Apoer2 and
Vldlr, and reeler mice (Trommsdorff et
al., 1999
) (for a review, see
Herz and Bock, 2002
). Upon
reelin binding to Apoer2/Vldlr receptors the cytosolic adapter protein mouse
disabled 1 (Dab1) is phosphorylated by Src family kinases
(D'Arcangelo et al., 1999
;
Howell et al., 1999
;
Hiesberger et al., 1999
;
Bock and Herz, 2003
;
Arnaud et al., 2003
) and
downstream signaling is thought to affect neuronal migration via cytoskeletal
changes (Howell et al., 1997
;
Rice et al., 1998
;
Hiesberger et al., 1999
;
Hammond et al., 2001
;
Beffert et al., 2002
). This
signaling pathway is supported in vivo since the phenotype of the
Dab1-deficient mice scrambler and yotari
(Sweet et al., 1996
;
Goldowitz et al., 1997
;
Gonzalez et al., 1997
;
Sheldon et al., 1997
;
Ware et al., 1997
;
Yoneshima et al., 1997
) and
the mice carrying a targeted deletion of the Dab1 gene
(Howell et al., 1997
)
corresponds to the reeler phenotype.
Importantly, however, VZ cells that are mostly composed of radial glia as
described above express Vldlr, Apoer2 and Dab1
(Sheldon et al., 1997;
Trommsdorff et al., 1999
;
Magdaleno et al., 2002
;
Benhayon et al., 2003
;
Luque et al., 2003
) and
phosphorylate Dab1 when stimulated with reelin
(Magdaleno et al., 2002
;
Benhayon et al., 2003
). Thus,
there is strong evidence that precursor cells, including radial glia, have the
prerequisite to directly perceive reelin signals. Indeed, misexpression of
reelin under control of the CNS-specific nestin-enhancer in VZ cells of reeler
mice leads to a partial rescue of the reeler phenotype
(Magdaleno et al., 2002
),
consistent with a potentially direct effect of reelin onto VZ cells. Moreover,
radial glial cells preferentially adhere to reelin-containing substrates in
vitro, an effect that also requires Dab1
(Förster et al., 2002
;
Frotscher et al., 2003
). These
results prompt the suggestion that reelin might also directly act on radial
glial cells. Here we tested this suggestion directly by isolating radial glial
cells as described previously (Malatesta
et al., 2000
; Malatesta et
al., 2003
; Heins et al.,
2002
). Moreover, we address the specific role of reelin signaling
to radial glial cells in vivo in reeler mice and combine this analysis with
complementary gain-of-function experiments by addition of reelin to radial
glial cells in vitro.
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Materials and methods |
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Cell dissociation and reelin conditioned medium
Acutely dissociated cells were prepared as described previously
(Hartfuss et al., 2001).
Conditioned medium was collected from 293 cells expressing either reelin or a
control plasmid containing GFP (clone pCrl) (see
D'Arcangelo et al., 1997
;
Förster et al., 2002
)
(kindly provided by T. Curran, St. Jude Childrens Hospital, Memphis Tennessee,
USA) 2 days after culturing in medium containing 10% fetal calf serum (FCS;
Sigma) or in chemically defined medium (see
Malatesta et al., 2000
). The
difference in reelin content was confirmed by western blotting using the mABs
E4 or G10 directed against reelin (De
Bergeyck et al., 1998
) (Fig.
5E).
|
BrdU labelling
Pregnant mice were injected intraperitoneally 1 hour prior to hysterectomy
with 5-bromo-2-deoxyuridin (BrdU, 5 mg in PBS per 100g body weight). In vitro,
BrdU was added at a final concentration of 10 µM either for the whole
culture period to label all dividing cells, or, to detect cells in S phase,
for only 1 hour (after 10 hours culturing of cells without BrdU, BrdU was
added for 1 hour, then BrdU was removed by several washes and cultures were
further incubated in BrdU-free medium for 11 hours).
DiI-labelling and 3D-reconstruction
The lipophilic dye
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate (DiI, Molecular Probes) was injected as ethanol/sucrose suspension
in the lateral ventricle of embryonic brains or the lumen of the spinal cord
(C57BL6/J, E12-16) ex vivo and stored in 2% paraformaldehyde in PBS for 10-20
days at room temperature resulting in a complete labelling of cell membranes.
Frontal vibratome sections (150 µm) were cut and analysed by confocal laser
scanning microscopy (CLSM). Series of single optical section images (1
µm) were used to assemble a 3D-reconstruction of the brain slice using
ImarisTM-program (Bitplane AG, Switzerland) that were used for the
morphological analysis of DiI-labeled cells as described in
Fig. 3.
|
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Results |
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|
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Notably, the proportion of VZ cells with a long radial process was significantly decreased in the reeler cortex (to 57% of the wild-type value; P<0.01; Fig. 3C,D) and the 'club shaped' population, characterized by a complete absence of a basal radial process, constituted the largest subpopulation of VZ cells in the reeler cortex (Fig. 3C,D). Even the population with 'short' basal processes ending underneath the cortical plate was reduced in the reeler cortex to less than half of their proportion in wild-type cortex (Fig. 3D), suggesting severe problems in radial fiber extension or maintenance in the absence of reelin. Interestingly, radial glial cells of the GE were not altered in their morphology in the reeler mutant (Fig. 3D), consistent with their normal Blbp-immunoreactivity (Fig. 1C-D''). Taken together, these results show a cortex-specific decrease in Blbp and radial processes in reeler mutant radial glia.
Proliferation and neuronal differentiation in the reeler
telencephalon
As the contact to the basement membrane is thought to influence cell
polarity and thereby cell proliferation and fate
(Huttner and Brand, 1997), we
examined these aspects in the reeler mutant cortex where fewer radial glial
cells are in contact with the basement membrane and also contain reduced
levels of Blbp that might affect these aspects
(Hartfuss et al., 2001
). We
first analyzed the number of mitotic cells in the VZ and the subventricular
zone (SVZ) by PH3-immunohistochemistry, but no significant difference was
detected between E14 wild-type and reeler littermates [PH3+cells/section,
wild-type cortex VZ 54±2 (n=804), SVZ 31±2
(n=463); reeler cortex VZ 49±2 (n=985), SVZ
23±2 (n=456)]. Regarding to the orientation of cell division,
no differences could be observed between wild-type and reeler mutant
littermates [wild-type cortex: angle to the ventricular surface is 90-60°,
71±14%; 60-30°, 6±6%; and 30-0°, 23±12%
(n=111)] [reeler cortex: 90-60°, 70±8%; 60-30°,
7±9%; and 30-0°, 24±9% (n=97)]. Thus, no changes in
the total number of precursors or the orientation of their cell division could
be detected in the reeler mutant cortex. In addition the progeny of radial
glial cells seemed not affected by the absence of reelin as the
immunoreactivity of the neuronal marker ß-tubulin-III was closely
comparable between wild-type and reeler mice
(Fig. 4A), suggesting that
neurogenesis also occurs normally in the absence of reelin. Because, however,
the precursors with long radial processes were reduced by 14% in the reeler
cerebral cortex, one might expect only a small reduction in the number of
neurons generated. To detect such small alterations in the number of neurons
we used FACS analysis of crosses between reeler and Tau::EGFP-mice that
contain the EGFP gene in the Tau locus and hence all neurons are green
fluorescent (Tucker et al.,
2001
; Heins et al.,
2002
). However, the number of neurons is identical in wild-type
and reeler mutant cortex at E14 (Fig.
4B). Thus, neither the lack of reelin nor the decrease in Blbp and
the decrease in pial attachment seems to exert a detectable effect on cell
proliferation or the generation of neuronal progeny by radial glial cells.
|
However, the increase in Blbp-positive cells and mRNA could also result
from a selective increase in the proliferation of the Blbp-expressing
subpopulation of precursors. To test this possibility, cells were cultured in
the presence of the DNA-base analogue BrdU in reelin-conditioned or control
medium (see Materials and methods for details). However, the analysis of the
labelling index (LI=BrdU+cells/Ki67+ precursors)
(Hartfuss et al., 2001) of
Rc2-single- and Rc2/Blbp-double-immunoreactive cells showed no increase in the
proliferation of Blbp-positive cells in reelin-conditioned medium, either when
BrdU was present in the medium for 24 hours [control LI Rc2 0.89±0.05,
LI Rc2/Blbp 0.96±0.03 (n=607); Rln-medium LI Rc2
0.93±0.1, LI Rc2/Blbp 0.83±0.04 (n=617)] or when BrdU
was added for only 1 hour [S-phase labelling control LI Rc2 0.37±0.17,
LI Rc2/Blbp 0.45±0.09 (n=595); Rln-medium LI Rc2
0.47±0.12, LI Rc2/Blbp 0.53±0.17 (n=603)]. Moreover, we
could not detect any significant effect of reelin on neuronal differentiation
and the overall population of precursors [ß-tubulin-III-positive neurons
after 24 hours in control medium: 53±5% (n=411); in
reelin-conditioned medium: 61±1% (n=425); P>0.2].
Taken together, these data show that the increase in Blbp-positive cells after
addition of reelin is not due to alterations in cell division or
differentiation but is caused by specific upregulation of Blbp protein and
mRNA in radial glial cells that were formerly Blbp negative.
Reelin increases the length of radial glia processes in vitro
As we found a correlation between the decrease of Blbp and radial processes
in radial glia of the reeler cerebral cortex, we next tested whether the
addition of reelin in vitro is also able to influence the process extension of
radial glial cells. When cells were cultured in control medium, about 50% of
precursor cells had extended processes of more than a cell diameter length 24
hours after dissociation (Fig.
5F,G). The vast majority of these cells had a bipolar morphology
and virtually no precursors acquired a multipolar morphology. Interestingly,
the proportion of precursors with a bipolar morphology in vitro (53%) closely
resembles the proportion of VZ cells with a long radial morphology labeled in
vivo (57%). In reelin-conditioned medium, however, the number of cells with
processes increased significantly to 80-90% (P<0.01) and no
significant difference was detectable between precursors from wild-type or
reeler mutant cortex (Fig.
5F,G), consistent with a full rescue of the deficits in process
length of reeler mutant radial glia in vitro. Taken together, these results
show that reelin addition affects Blbp-immunoreactivity and the extension of
radial processes from radial glial cells.
Direct signaling of reelin to radial glial cells
To examine whether the upregulation of Blbp by reelin is mediated by direct
signaling to radial glia or mediated indirectly via the neurons present in the
cultures, we used two approaches to reduce the number of neurons. First we
isolated the non-fluorescent cells from the E14 cortex of the Tau::EGFP mouse
line that contains GFP specifically in all neurons (see above) (see
Tucker et al., 2001;
Heins et al., 2002
). Selection
of GFP-negative cells reduced the proportion of neurons from 57%±3
(n=767) in unselected cells to 25% ß-tubulin-III-immunoreactive
cells analyzed 2 hours after sorting and plating (n=123).
Interestingly, the increase in Blbp-immunoreactive cells after reelin addition
was not reduced but even higher in these cultures despite the reduced number
of neurons (Fig. 6A',
compare to Fig. 5B). However,
because a quarter of all cells were still neurons in these cultures, we
further decreased their proportion by positively sorting radial glial cells
from the hGFAP-GFP-mouse line that contains GFP in radial glial cells as
described previously (Malatesta et al.,
2000
; Heins et al.,
2002
). When GFP-positive cells were isolated by FACS
(Fig. 6B), sorted cells were
almost all Rc2-positive and only 13%±7 (n=197) were
ß-tubulin-III-immunoreactive 2 hours after sorting. However, despite the
strong reduction of neurons in these cultures Blbp-immunoreactivity could
still be significantly (P<0.04) increased by reelin-conditioned
medium (Fig. 6B'),
strongly suggesting that Blbp upregulation is mediated by direct signaling to
radial glial cells.
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Discussion |
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Interestingly, the lack of reelin seems to affect only a subpopulation of
radial glial cells, as some radial glial cells still manage to extend long
radial processes also in the reeler mutant and establish contacts with the
basement membrane forming a glia limitans that is only partially disrupted (N.
Haubst and M.G., unpublished) (Gadisseux
and Evrard, 1985). From the tight correlation between Blbp
regulation and radial process extension that we observed in this work, we
would hypothesize that the radial glial cells with normal process outgrowth
and anchorage were those that still contain Blbp even in the absence of
reelin, suggesting that reelin signaling is dispensable for some radial glia
to express Blbp and elongate a radial process. Indeed, Blbp-positive radial
glial cells in other CNS regions, such as the GE and the spinal cord, neither
require reelin signaling, as their morphology and Blbp content were not
affected in the reeler mice, nor react to reelin addition in vitro. This is in
pronounced contrast to the significant Blbp upregulation and radial process
extension evoked by reelin in cortical radial glia in vitro. These results
therefore suggest that several pathways exist that regulate radial glia
process extension and Blbp content. Indeed, a variety of mostly
uncharacterized factors have been proposed to act on process extension of
radial glial cells that are released either by neurons
(Hunter and Hatten, 1995
;
Anton et al., 1997
;
Rio et al., 1997
;
Hasling et al., 2003
) or by
cells located in the MZ (Soriano et al.,
1997
; Super et al.,
2000
) in different brain regions. Because several of these factors
also seem to act in the cerebral cortex, several signaling pathways apparently
converge to regulate radial glia process extension, consistent with the loss
of some, but not all, long radial processes in the reeler cortex. However, the
clear defects of radial glia morphology in the reeler cerebral cortex and
hippocampus (Förster et al.,
2002
; Weiss et al.,
2003
) underline the importance of this signaling pathway. These
defects had previously been interpreted as premature transformation of radial
glial cells in the reeler mutants
(Hunter-Schaedle, 1997
). Our
data do not support this interpretation. First, the normal increase in Blbp
during development in wild-type cortex is decreased from its onset in the
reeler mutant cortex rather than occurring prematurely. Second, we did not
observe the decrease in Rc2-immunoreactivity (see
Fig. 1) or the premature
increase in GFAP-immunoreactivity (data not shown) in reeler mutant radial
glial cells described previously
(Hunter-Schaedle, 1997
).
Moreover, the increase in precursors without any basally oriented radial
process in the reeler mutant cortex described here is notably different from
the bi- or multipolar glial precursors leaving the VZ as observed in mouse
mutants with premature glial differentiation
(Nieto et al., 2001
).
Similarly, the normal proliferation and neurogenesis as examined here in
detail for the reeler cortex also does not support the notion of premature
gliogenesis. Finally, reeler mutant mice exhibit also in the cerebellum
several defects in glial differentiation, including the reduction of Glast
(Ghandour et al., 1981
;
Benjelloun-Touimi et al., 1985
;
Fukaya et al., 1999
). Taken
together, these data suggest that reelin signaling is required for some
aspects of radial glia and astrocyte differentiation.
Radial glia defects and their role as precursors
Importantly, the radial glia defects of reeler mutants differ from those of
other mouse mutants, such as the Pax6 mutant where Glast, but not
Blbp, is downregulated (Götz et al.,
1998; Heins et al.,
2002
). Reelin-secreting cells are even increased in number in the
MZ of the Pax6 mutant cortex
(Stoykova et al., 2003
). In
contrast to the profound defects in the precursor function of radial glial
cells in this cortex (Heins et al.,
2002
; Estivill-Torrus et al.,
2002
), no such defects were detected in the cortex of reeler
mutant mice. The neurochemical and morphological defects of reelin-deficient
radial glial cells occur in 50-15% of the precursor pool (Figs
1,
2,
3), but not even small changes
in neurogenesis or cell proliferation could be detected implying that neither
Blbp nor the long radial process is necessary for the precursor function of
radial glial cells.
Radial glia defects and neuronal migration
In contrast to the normal cell fate and cell proliferation, radial glia
defects are likely to contribute to the defects in radial cell migration
observed in the reeler cortex. In line with this suggestion is the
region-specific correlation of radial glial defects and aberrant radial
migration in the reeler mutant mice. Indeed, radial glia morphology or Blbp
content is normal in the spinal cord of reeler mice (data not shown) as is
radial cell migration. Interestingly, only the tangential migration of
preganglionic neurons is affected in the spinal cord of reelin-deficient mice
(Yip et al., 2000;
Phelps et al., 2002
). Thus,
the effect of reelin on tangential cell migration seems to be mediated in a
radial glia independent manner, potentially by signaling directly to the
migrating neurons. With regard to radial cell migration, however, the radial
glia process seems essential both for glia-guided neuronal migration
(Rakic, 1972
), as well as the
somal translocation of neurons generated from radial glia maintaining their
basal processes (Morest, 1970
;
Miyata et al., 2001
;
Nadarajah et al., 2001
). As
the cortex constantly increases its thickness during development, both modes
of radial cell migration depend crucially on radial process extension.
Notably, a recent model explains the defects in cortical layering of reeler
mutants solely by the defects in radial process of neurons migrating by somal
translocation (Luque et al.,
2003
).
Direct reelin signaling to radial glia
Our data suggest that these defects in radial glial cells are mediated by
direct signaling of reelin to radial glial cells. We show that radial glial
cells purified from the cerebral cortex react to reelin addition by
upregulation of Blbp. The almost complete absence of neurons in vitro even
enhanced this effect compared with cultures with many neurons. These data also
argue against an additional indirect effect on Blbp mediated via neuronal
signaling. Taken together with the early onset of the defect in Blbp in reeler
mutant cortex, these data suggest that the radial glia phenotype in reeler
mice is mostly due to the lack of direct signaling to radial glia, rather than
some indirect effects, e.g. aberrant neuronal migration. It has been suggested
previously that neuronal attachment to radial glia positively influences their
Blbp content (Feng et al.,
1994). However, the attachment of neurons to radial glia is rather
increased in the reeler cortex (e.g.
Caviness et al., 1988
) and can
thus not explain the decrease in Blbp-positive cells. Taken together, these
results therefore favor a direct regulation of Blbp by reelin signaling to
radial glia in vivo as demonstrated directly in vitro.
In vivo, reelin most probably acts on radial glia in the MZ where their
processes are in close contact to the reelin-secreting cells (see
Fig. 1). Because
reelin-secreting cells are only a small subpopulation of cortical neurons,
their secretion of reelin into the culture medium in vitro is negligible and
hardly detectable in western blot analysis of the culture medium (data not
shown). Accordingly, the increase of Blbp-positive cells during development in
vivo (Hartfuss et al., 2001)
is halted in vitro where no change in Blbp-positive cells can be observed
during 24 hours. However, the high reelin content in medium conditioned by a
reelin-secreting cell line results in Blbp upregulation and the extension of
radial processes. This in vitro model might therefore mimic the situation in
the MZ, where reelin concentrations are high, and might influence process
extension or maintenance possibly via the regulation of Blbp. Consistent with
this suggestion is the close correlation of the appearance of
reelin-(Stoykova et al., 2003
)
and Blbp-immunoreactive cells in wild-type cortex
(Hartfuss et al., 2001
) at a
time when the cortex starts to thicken and radial glia need to extend their
processes. At postnatal stages, the transformation of radial glia into
astrocytes coincides with the degeneration of reelin-secreting cells in layer
1 (Super and Uylings,
2001
).
Experiments in which reelin is misexpressed in nestin-positive cells (most
of which are radial glia as described above) further support the direct
signaling of reelin to radial glial cells
(Magdaleno et al., 2002).
Reelin synthesis in VZ precursors is sufficient to rescue some, but not all,
migrational defects of the reeler mutant cortex
(Magdaleno et al., 2002
).
These data therefore suggest either that direct signaling to neurons is
responsible for the effects not rescued in these transgenic mice, or that
reelin has to be deposited in the MZ, either to be highly concentrated in the
extracellular milieu present there or to provide a directional signal to
radial glia, e.g. to stabilize their contact to the basement membrane. As we
have shown that reelin signaling acts via the same pathway (via the adaptor
protein Dab1) in radial glia as it does in neurons, one may think of
experimentally addressing the relative contribution of reelin signaling to
radial glia and neurons by, for example, a neuron-specific Cre-mediated
knockout of Dab1. However, when most neurons migrate by somal translocation
(see Luque et al., 2003
), this
issue becomes semantic. In the cases when the basally generated neuron
inherits the radial process from its mother radial glia
(Miyata et al., 2001
) and then
migrates by somal translocation, reelin always signals to the same radial
process, first the one of the radial glia mother cell, and later to the
process of the differentiating and migrating neuron. However, recent
experiments suggest that somal translocation might dominate only during early
stages of cortical development (Nadarajah
et al., 2001
) and become less frequent at later stages
(Weissman et al., 2003
). The
most crucial next experiment would therefore be to examine the mode of cell
migration in the reeler mutant cortex, whether it is really predominantly
somal translocation that is stalled in the absence of reelin (or Dab1). Taken
together, these results shed new light on the migrational defects of the
reeler mutation and suggest a crucial role of a direct signaling of reelin to
radial glial cells.
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ACKNOWLEDGMENTS |
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