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The Journal of Neuroscience, January 1, 2003, 23(1):203-212
Abnormal Development of Forebrain Midline Glia and Commissural
Projections in Nfia Knock-Out Mice
Tianzhi
Shu1,
Kenneth G.
Butz2,
Celine
Plachez1,
Richard M.
Gronostajski2, and
Linda J.
Richards1
1 Department of Anatomy and Neurobiology, and the
Program in Neuroscience, The University of Maryland, Baltimore, School
of Medicine, Baltimore, Maryland 21201, and 2 Department of
Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation,
and Department of Biochemistry, Case Western Reserve University,
Cleveland, Ohio 44195
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ABSTRACT |
Nuclear factor I (NFI) genes are expressed in multiple
organs throughout development (Chaudhry et al., 1997 ; for review, see Gronostajski, 2000). All four NFI genes are expressed in embryonic mouse brain, with Nfia, Nfib, and
Nfix being expressed highly in developing cortex
(Chaudhry et al., 1997). Disruption of the Nfia gene
causes agenesis of the corpus callosum (ACC), hydrocephalus, and
reduced GFAP expression (das Neves et al., 1999). Three midline structures, the glial wedge, glia within the indusium griseum, and the
glial sling are involved in development of the corpus callosum (Silver
et al., 1982; Silver and Ogawa, 1983; Shu and Richards, 2001). Because
Nfia /
mice show glial abnormalities and ACC, we asked whether defects in
midline glial structures occur in
Nfia/
mice. NFI-A protein is expressed in all three midline populations. In
Nfia/,
mice sling cells are generated but migrate abnormally into the septum
and do not form a sling. Glia within the indusium griseum and the glial
wedge are greatly reduced or absent and consequently Slit2 expression
is also reduced. Although callosal axons approach the midline, they
fail to cross and extend aberrantly into the septum. The hippocampal
commissure is absent or reduced, whereas the ipsilaterally projecting
perforating axons (Hankin and Silver, 1988; Shu et al., 2001) appear
relatively normal. These results support an essential role for midline
glia in callosum development and a role for Nfia in the
formation of midline glial structures.
Key words:
glial wedge; glial sling; glial tunnel; corpus
callosum; anterior commissure; hippocampal commissure; Slit2
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Introduction |
Midline glia are associated with the
development of commissural projections in the mammalian brain (Katz et
al., 1983; Guillery and Walsh, 1987; Godement and Mason, 1993; Silver,
1993; Silver et al., 1993; Reese et al., 1994; Marcus et al., 1995;
Cummings et al., 1997; Pires-Neto et al., 1998). At the optic chiasm,
specialized midline cells and glia regulate which axons project
ipsilaterally and contralaterally by differentially guiding axons at
the midline (Marcus et al., 1995; Wang et al., 1995). In the
development of the corpus callosum, several midline populations, the
glial sling, the glial wedge, and glia within the indusium griseum,
have been implicated in callosal axon guidance (Silver et al., 1982;
Silver and Ogawa, 1983; Shu and Richards, 2001).
In both lesioning and grafting experiments, Silver and colleagues
(Silver et al., 1982; Silver and Ogawa, 1983) showed that the glial
sling was important for the formation of the corpus callosum. Two other
midline populations, the glial wedge and glia within the indusium
griseum, may also be important for callosal formation (Shu and
Richards, 2001). In collagen gels, glial wedge explants secrete a
short-range diffusible repellent/growth suppressive molecule, and in
organotypic slices, reorientation of the glial wedge and glia within
the indusium griseum causes callosal axons to turn away from the
midline (Shu and Richards, 2001). These results indicate a role for
these midline populations in the development of the corpus callosum.
A large number of single gene mutations in mice result in ACC, yet in
many cases it is not known how these genes function to prevent the
formation of this commissure. In an effort to understand some of the
critical developmental events required for callosal formation in
vivo, we have investigated whether the development of midline
structures may be disrupted in the Nfia mutant, which was
reported previously to exhibit both agenesis of the corpus callosum and
a reduction in glial fibrillary acidic protein (GFAP) expression (das
Neves et al., 1999). Nfia belongs to the nuclear factor I (Nfi) family of transcription factors that function both in
the regulation of adenoviral DNA replication and in viral and cellular
gene expression, including the control of olfactory-specific genes
(Nagata et al., 1982, 1983; Gronostajski et al., 1985; Hennighausen et
al., 1985; Leegwater et al., 1985; Nowock et al., 1985; Baumeister et
al., 1999; Behrens et al., 2000).
Given the role of Nfia in GFAP expression and the acallosal
phenotype of Nfia knock-out animals, we hypothesized that
midline glia known to be involved in callosal development may be
disrupted in Nfia mutant mice. We found that Nfia
is expressed in midline glial structures and that the development of
these structures is severely impaired in
Nfia/
mice. These data indicate that Nfia may regulate callosal
development by regulating the development of midline glia.
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Materials and Methods |
Animals. Animals used for NFI-A immunohistochemistry
were wild-type C57BL/6J mice (The Jackson Laboratory, Bar Harbor,
ME) bred on site at The University of Maryland, Baltimore animal
facility under the care and approval of the accredited University of
Maryland, Baltimore Institutional Animal Care and Use Committee.
Nfia/
animals used in all other experiments were generated and bred at The
Lerner Research Institute (Cleveland, OH).
Nfia/
mice were generated using E14-1 embryonic stem cells (derived from
strain 129P2) (Hooper et al., 1987; Festing et al., 1999) and
backcrossed onto a random-bred Swiss background (strain Tac:N:NIHS-BC) as described previously (das Neves et al., 1999). The
Nfia/
allele was then backcrossed onto the C57BL/6NTac strain (Taconic, Germantown, NY) for >10 generations, and all embryonic and postnatal mice used in these experiments were derived from these mice. Unlike a
number of 129 strains (Livy and Wahlsten, 1991, 1997), C57BL/6 mice do
not display ACC. Furthermore, no defects in commissural tract formation
were detected in wild-type or heterozygous Nfia animals on a
C57BL/6 background; therefore, the phenotype described here is
independent of any preexisting strain affect. To obtain timed-pregnant
females, male and female mice were placed together overnight. Females
were weighed and inspected the following morning for the presence of a
vaginal plug. If present, this day was designated embryonic day 0. Heterozygous
Nfia+/ mice
were bred to obtain litters of (+/+),
(+/), and
(/)
mice that were in a roughly Mendelian ratio. Embryos were genotyped by
PCR according to the protocol described previously (das Neves et al.,
1999). On the required gestational day, embryos were either immersion-fixed or perfused with 4% paraformaldehyde [for
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) labeling]. The heads were then removed and stored in 4%
paraformaldehyde until use. Fixed heads were transported from Cleveland
to Baltimore for processing.
Immunohistochemistry. Wild-type brains stained for NFI-A
were perfused with 0.9% saline followed by a mixture of 4%
paraformaldehyde and then postfixed in 4% paraformaldehyde at 4°C
until sectioning. Heads from Nfia litters were
immersion-fixed in 4% paraformaldehyde at 4°C until the brains were
removed for sectioning. Before sectioning, the brains were blocked in
3% noble agar and then sectioned at 50 µm on a Vibratome (Leica,
Deerfield, IL). Immunohistochemistry was performed in a manner similar
to that described previously (Shu et al., 2000). Sections of
acrolein-fixed brains were first incubated in 1% sodium borohydride
(w/v) (Sigma, St. Louis, MO) PBS, pH 7.4, for 10 min to remove the
acrolein and then processed in an identical manner to the brains fixed
only in 4% paraformaldehyde. Sections were washed three times in PBS,
blocked in 2% normal goat serum (v/v) (Jackson ImmunoResearch
Laboratory, West Grove, PA) and 0.2% Triton X-100 (v/v) (Sigma) in PBS
for 2 hr, and then incubated in primary antibody [rabbit anti-NFI-A,
at either 1:1000 or 1:75,000 (affinity-purified antibody, catalog
#16111460; Geneka Biotechnology, Montreal, Canada); rabbit anti-GFAP,
1:30,000 (Dako, Glostrup, Denmark); rabbit anti-neurofilament M
C-terminal, 1:10,000 (Chemicon, Temecula, CA); mouse anti-neuronal
nuclei (NeuN), 1:3000 (Chemicon)] overnight. On day 2, sections were
washed three times in PBS and then incubated in biotinylated goat
anti-rabbit secondary antibody (1:600; Jackson ImmunoResearch
Laboratory) for 1 hr. After three washes in PBS, sections were
incubated in avidin-biotin solution (1:500, Vector Laboratories,
Burlingame, CA) for 1 hr, followed by three washes in PBS. Sections
were then immersed in a nickel/3,3' diaminobenzidine (DAB) chromogen
solution (2.5% nickel sulfate and 0.02% DAB in 0.175 M sodium acetate) activated with 0.01% (v/v)
H2O2 until a dark
purple/black precipitate formed. Sections were washed in PBS, mounted,
and coverslipped in DPX mounting medium (Electron Microscopy
Services). For brain lipid binding protein (BLBP) (Feng et al.,
1994; Kurtz et al., 1994) immunohistochemistry, brains were perfused in
4% paraformaldehyde and vibratome sectioned and blocked as described
above. BLBP primary antibody (polyclonal; a gift from Dr. N. Heinz,
Rockefeller University, New York, NY) was used at 1:8000 (diluted in
blocking solution) overnight, washed three times, and then incubated
with a donkey anti-rabbit Cy3-conjugated antibody (1:400; Jackson
ImmunoResearch Laboratories). For double immunohistochemical staining
of NFI-A or BLBP and GFAP, sections were stained as described above for the first primary antibody (anti-NFI-A or anti-BLBP) and then incubated
in donkey anti-rabbit Cy2- or Cy3-conjugated antibody (1:400; Jackson
ImmunoResearch Laboratories). Sections were then reblocked
in PBS with 2% normal donkey serum and 0.2% Triton X-100 (Sigma) and
then incubated in the second primary antibody (1:1000, rabbit-anti-GFAP; Dako) overnight. Sections were washed as described for single labeling and then incubated in a donkey anti-rabbit Cy2- or
Cy3-conjugated secondary antibody (1:400; Jackson ImmunoResearch Laboratories) for 2 hr. The sections were then washed,
mounted, and coverslipped with polyvinyl alcohol
(PVA)/1,4-diazabicyclo(2.2.2)octane (DABCO) mounting medium
[consisting of 9.6 gm PVA (Sigma)/DABCO (Sigma) in a solution
containing 24 gm glycerol, 48 ml 0.2 M Tris-HCl, pH 8-8.5, and 24 ml H2O] for confocal microscopy.
In situ hybridization. Digoxigenin was used to
label Tbr-1 probes (a gift from Dr. J. L. Rubenstein,
University of California, San Francisco, San Francisco, CA).
Nonradioactive in situ hybridization was performed on 45 µm floating Vibratome sections. Sections were washed in PBS, pH 7.4, and acetylated for 10 min with 0.25% acetic anhydride in 1%
triethanolamine solution. Two washes in PBS for 5 min were followed by
an equilibration in 2× standard saline citrate (SSC) for 5 min.
Sections were prehybridized overnight at room temperature in 50%
formamide, 5× Denhardt's solution, 5× SSC, and 250 µg/ml
baker's yeast tRNA. The hybridization mixture was the same as the
prehybridization mixture but also contained 3 ng/ml DIG-labeled
riboprobe. Hybridization was performed at 60°C overnight. Sections
were washed at 60°C for 5 min in 5× SSC, 1 min in 2× SSC, 30 min in
50% formamide containing 0.2× SSC, and 5 min in 0.2× SSC. Sections
were then rinsed in Tris-buffered saline (TBS; 100 mM Tris-HCl, pH 7.5, and 150 mM NaCl) for 5 min and incubated for 1 hr in 1%
blocking reagent (Roche, Hertforshire, UK) in TBS. For immunodetection,
sections were incubated for 1 hr with an anti-DIG Fab fragment
conjugated to alkaline phosphatase (anti-DIG-AP; Roche) at
a dilution of 1:5000. Sections were then rinsed twice for 15 min in TBS
and once in 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2.
The color reaction was performed with nitroblue tetrazolium chloride
and 5-bromo-4-chloro-3-indolylphosphate (Roche). Sections were rinsed
in 10 mM Tris-HCl, pH 8.0, 5 mM EDTA (TE), mounted on glass slides, and
coverslipped with Hydromount (National Diagnostics, Atlanta, GA).
Controls consisted of sections subjected to the complete in
situ hybridization procedure, but with no probe or hybridized with
a Tbr-1 sense probe. These exhibited no specific hybridization signal.
For Slit2, Robo1, and Robo2 in situ hybridization, probes
were made from 1 µg DNA (plasmids were gifts from Dr. M. Tessier-Lavigne, Stanford, CA), radiolabeled with
[S35]UTP (40 mCi/ml). Probes were
purified by DNase digestion and precipitated in 0.6 M NH4OAC and 100% ethanol.
Slit2 and Robo1 probes were hydrolyzed, but the Robo2 probe did not
need to be hydrolyzed. For hydrolyzing the probes, 20 µl of 40 mM sodium bicarbonate and 30 µl of 60 mM sodium carbonate were added to the probes and
then incubated at 65°C for 30 min. Probes were again precipitated in
NH4OAC and ethanol and then counted in a scintillation counter. A concentration of 400,000-600,000 cpm/µl was
generally obtained from the above procedures. Cryostat sections (10 µm) were pretreated with 4% paraformaldehyde for 20 min and 2× SSC
at 65°C for 30 min. This was followed by a 10 min treatment with
Proteinase K (20 µg/ml in TE) and fresh acetylation mix with 3.25 ml
triethanolamine and 0.7 ml acetic anhydride in 250 ml DEPC water.
Sections were dehydrated through a 70-100% ethanol. Probes (50,000 cpm/µl) were diluted in the hybridization buffer with 50% deionized
formamide, 10% dextran sulfate, 1× Denhardt's solution, 0.3 M NaCl, 10 mM Tris, pH 7.5, 10 mM sodium phosphate, pH 6.8, 5 mM EDTA, 25 mM DTT, and 50 mM  -mercaptoethanol. Hybridization was
performed at 55°C overnight. Sections were washed in 5× SSC and 20 mM -mercaptoethanol at 55°C for 30 min, and
then a solution of 2× SSC, 50% formamide, and 20 mM -mercaptoethanol at 65°C for 30 min at
room temperature for an additional 1 hr. Sections were further washed
in TE, RNase A, and SSC before being dehydrated through ethanol series
with NH4OAC. Slides were dipped in NTB-2 emulsion
(Kodak) and stored at room temperature for 4 weeks before being
developed in D-19 and fixer (Kodak).
1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate and
4-(4-(dihexadecylamino)styryl)-N-methyl-pyridinium iodide labeling of axons. Tract tracing experiments were performed using a 3% solution of
4-(4-(dihexadecylamino)styryl)-N-methyl-pyridinium iodide
(DiA) (Molecular Probes, Eugene, OR) and/or a 10% solution of
1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes) in dimethylformamide (Sigma). Brains from
embryonic day (E) 15, E17, E18, and postnatal day 0 (P0) Nfia litters were perfused with 0.9% saline followed by 4%
paraformaldehyde and then postfixed in 4% paraformaldehyde until
injection. Embryos were genotyped by PCR as described previously (das
Neves et al., 1999). Dye injections were performed using a fine-tipped
(pulled) glass micropipette attached to a Picospritzer (Parker
Instrumentation, Fairfield, NJ). DiI injections were made in the cortex
just lateral to the midline from rostral to caudal of one hemisphere,
or at the midline of a hemisected brain (see Fig. 1H)
to label callosal axons. In some brains, a coronal cut was made
caudally through the hippocampus to expose the structure. DiA was then
injected into the hippocampus to label the fornix and the hippocampal
commissural axons. Brains were stored in the dark at room temperature
for at least 4 weeks to allow DiI and DiA transportation. Brains were blocked in 3% noble agar and cut at 50 µm on a Vibratome. The sections were then mounted and coverslipped with PVA/DABCO mounting medium for confocal microscopy as described above. Some sections (see
Fig. 1H) were counterstained with 10 nm Sytox green
(Molecular Probes) in DMSO for 1-2 hr and then washed three times in
PBS before mounting and analysis.
Preparation of figures. Bright-field images were scanned
directly into Photoshop (Adobe) on an upright microscope (Leica) equipped with a digital scanning camera (Phase One). All fluorescent images were collected on a scanning laser confocal microscope (Olympus
Fluoview) and then transferred to Photoshop (Adobe) for processing.
Collages were made of the images, and the appropriate labels were
added. No image processing was performed other than enhancing the
brightness and contrast and cropping the image. Figures were made on a
Macintosh G4 computer.
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Results |
Expression of NFI-A at the developing cortical midline
To characterize the expression of NFI-A protein, we performed
immunohistochemistry on developing mouse brain sections using a
polyclonal NFI-A antibody (Geneka Biotechnology, Montreal, Canada). This anti-peptide antisera is directed against a peptide epitope present only on NFI-A proteins and not present in NFI-B, -C, or -X
proteins. At E15, NFI-A was expressed within the ventricular zone and
the emerging cortical plate (Fig.
1A,B).
At E16 and E17, NFI-A expression was confined to the subplate and the
deeper layers of the neocortex (Fig. 1C,E). A
higher power view of the cortical plate at E17 shows that NFI-A
labeling is mostly confined to the subplate, layer 6, and possibly the
most ventral part of layer 5 (Fig. 1F). Very low
levels of expression were seen in the upper layers of the cortical
plate (Fig. 1F). To examine this expression further,
we performed in situ hybridization for Tbr-1, which labels the subplate and layer 6 (Hevner et al., 2001), on an adjacent section
(Fig. 1G). NFI-A and Tbr-1 overlapped in their expression patterns, indicating that by this criterion at least, NFI-A labeled both the subplate and layer 6. Most of the callosal axons arise from
neurons in layers 2/3 and 5 (Wise and Jones, 1976). To determine whether NFI-A was expressed in callosal neurons, we retrogradely labeled callosal neurons at E17 with DiI from an injection into the
corpus callosum at the midline (Fig. 1H). By
comparing the DiI labeling with NFI-A labeling, we found that most
DiI-filled cells were in regions that did not express NFI-A (Fig. 1,
compare F, H). This indicated that most of
the callosally projecting axons probably do not express NFI-A.
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Figure 1.
NFI-A expression in the developing mouse cortex.
Sections of wild-type (C57BL/6J) brains were processed for NFI-A
immunohistochemistry at E15 (A, B), E16
(C, D), and E17 (E,
F, H-K). At E15,
NFI-A is expressed in the dorsal telencephalon, including the
neocortical plate and the cingulate cortex, but stops at the
corticoseptal boundary (A, B). Cells within the region
of the glial wedge express NFI-A (B,
large arrow). At E16 and E17, expression became more
confined to the deeper layers of the cortex (C,
E) and is evident at the midline in the indusium griseum
(D, arrowhead), the glial sling
(D, small arrow), and the glial wedge
(D, large arrow). At higher power, NFI-A
labeling in the subplate and layer 6 (F) overlaps
with Tbr-1 labeling in an adjacent section (G).
Retrograde labeling of callosal neurons at E17 with DiI (counterstained
with Sytox green) in an area similar to F and
G shows that most of the callosally projecting neurons
are not in an area of high NFI-A labeling
(H). At E17, NFI-A is expressed in midline
glial populations (I, J',
K', green nuclei) double-labeled with
GFAP immunohistochemistry (I, J', and
K', red labeling in the cytoplasm). Cells
within the glial wedge (boxed region labeled
J) and the indusium griseum (boxed
region labeled K) express both NFI-A and
GFAP as seen in higher-power views of these regions (J'
and K', respectively). The glial sling is NFI-A positive
(D, small arrow) but GFAP negative
(I). Scale bar: (in K')
A, C, 600 µm; B,
D, 150 µm; I, 50 µm;
J', K', 20 µm; (in
H) E, 1000 µm;
F-H, 100 µm.
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At the cortical midline, NFI-A expression was observed within the
indusium griseum (Fig. 1D, arrowhead,
I,K,K'), the glial sling
(Fig. 1D, small arrow), and the glial
wedge cell bodies (Fig. 1B,D,
large arrows,
I,J,J'). Double staining
of NFI-A in the nucleus (green) and GFAP in the
cytoplasm (red) in the same section revealed many cells
labeled for both NFI-A and GFAP in the glial wedge and the indusium
griseum glia (Fig.
1I,J',K').
Abnormal development of midline glia in the Nfia
knock-out mouse
We used GFAP immunohistochemistry to examine the development of
the glial wedge and glia within the indusium griseum because this is a
reliable marker of these glial populations. Although it had been shown
previously that GFAP expression was greatly reduced in the
Nfia/
adult mice (das Neves et al., 1999), we found that GFAP was still expressed at embryonic stages and therefore could be used to identify these structures in the knock-out. In heterozygous
Nfia+/ mice
the glial wedge formed normally at E15 (Fig.
2A) but was not present
in
Nfia/
littermates (Fig. 2B). At E17 the glial wedge was
clearly evident on either side of the midline and underlying the
developing corpus callosum in heterozygote mice (Fig. 2C,
white arrow); however, only a few GFAP-positive processes
within the wedge were present in the
Nfia/
mice (Fig. 2D, white arrow), and the
corticoseptal boundary was not well defined. Glia within the indusium
griseum also formed normally by E17 in the heterozygotes (Fig.
2C, white arrowhead), but in the
Nfia/
mice some glia were present at the midline (Fig. 2D,
white arrowhead); however, it was not clear whether these
were displaced/malformed indusium griseum glia or, alternatively,
midline zipper glia (Silver et al., 1982, 1993). Glia within the
hippocampus, particularly the dentate gyrus and the fimbria, were
present in the
Nfia+/ mice
(Fig. 2E, white arrow) but were also
reduced in the
Nfia/
mice (Fig. 2F, white arrow). Finally, we
also examined the formation of the glial tunnel that surrounds the
anterior commissure (Katz et al., 1983; Silver et al., 1993; Cummings
et al., 1997; Pires-Neto et al., 1998). This structure is best seen in
sagittal sections. In Nfia
+/ mice the
glial tunnel formed normally with fibers surrounding the anterior
commissure (Fig. 2G, arrow). In
Nfia/
mice, however, the number of GFAP-expressing glial fibers was greatly
reduced, and the region occupied by the anterior commissure was much
thinner in the rostrocaudal axis (Fig. 2H,
arrow), although it appeared normal in the dorsoventral axis
(see Fig. 7H). These results suggest that
Nfia may participate in the development of glia in the
forebrain, perhaps by regulating GFAP expression.
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Figure 2.
Development of medial glial populations in
Nfia/
mice. At E15 the long radial glial-like processes of the glial wedge
are evident by GFAP staining in
Nfia+/
mice (A, arrow), but are not evident in
the knock-out (B), indicating a possible delay in
glial wedge development. At E17 the glial wedge (C,
arrow) and the indusium griseum glia (C,
arrowhead) are clearly evident in the heterozygote. In
the knock-out the glial wedge is greatly reduced (D,
arrow) and additional glia arise at the fusion point
between the two hemispheres (D,
arrowhead), but it is not clear whether these are
displaced indusium griseum glia or midline "zipper" glia, which are
thought to participate in midline fusion. In the hippocampus, a large
number of glial processes are evident, particularly in the dentate
gyrus and the fimbria (E, arrow) but are
greatly reduced in the knock-out (F,
arrow). In sagittal sections (G,
H), the glial tunnel is evident in
Nfia+/
mice but greatly reduced in the knock-out (H,
arrow). Insets in each figure show a
low-power view of each section. Scale bar: (in H)
A, B, 100 µm; C,
D, 150 µm; E, F, 70 µm; G, H, 10 µm.
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Given that Nfia may regulate GFAP expression, it may be that
there were glia present at the midline but not expressing GFAP. To
investigate this, we labeled sections with BLBP, a second marker for
these glial populations, using a polyclonal anti-BLBP antibody (a gift
from Dr. N. Heinz) (Feng et al., 1994) (Fig.
3). BLBP labels both structures in
wild-type littermates at E17 (Fig.
3B,H) as shown by double
immunohistochemistry for both GFAP and BLBP [Fig.
3C,I, yellow-labeled glial;
labeled with arrows (glial wedge) and arrowheads
(indusium griseum glia)]. Although some glial wedge processes were
present in
Nfia/
mice, there were fewer, and those that were present were shorter and
did not coalesce into the typical wedge-shaped structure (Fig. 3F, arrow). Labeling of the indusium griseum glia
with BLBP showed that this population was also absent or greatly
reduced in
Nfia/
mice compared with wild-type littermates (Fig.
3I,L, arrowheads). BLBP
expression was unchanged in other regions of the forebrain, including
the cortex and radial glial within the striatum (data not shown),
indicating that the reduced expression of BLBP in the glial wedge and
indusium griseum glia was unlikely to be caused by the regulation of
BLBP by Nfia.
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Figure 3.
GFAP and BLBP labeling of the glial wedge and
indusium griseum glia. To examine whether the reduction in GFAP
expression observed in the
Nfia/
mice reflected abnormalities in these glial structures, we
double-labeled sections at E17 with both GFAP (A,
D, G, J) and BLBP
(B, E, H,
K) and overlaid the images to examine labeling in
the same cells (C, F, I,
L). GFAP and BLBP labeled glial processes (not cell
bodies) in both the glial wedge (A and B,
respectively) and indusium griseum (G and
H, respectively) in wild-type animals. However, both the
glial wedge (C, F, I,
L, arrows) and indusium griseum glia
(C, I, L,
arrowheads) were either greatly reduced or absent in
Nfia/
mice. Glial wedge processes appeared shorter and failed to coalesce
into a wedge shape (F, arrow). Scale bar:
(in L) A-F, 150 µm;
G-L, 500 µm.
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Another structure associated with the development of the corpus
callosum is the glial sling (Silver et al., 1982). In rodents the glial
sling is not GFAP positive (Silver et al., 1993) but can be labeled
with the neuronal marker NeuN (Mullen et al., 1992), which, along with
morphological and anatomical criteria, is a reliable method for
identifying the sling (Preston et al., 2000; T. Shu and L. J. Richards, unpublished observation). As described previously by Silver
and colleagues (Silver et al., 1982), the sling is obvious
morphologically as a group of cell bodies at the midline between the
two cerebral hemispheres (Fig.
4A, white arrow). In
Nfia/
mice, at E17 the sling cells are generated but migrate into the septum
and do not form the sling structure (Fig. 4B,
white arrow). In more rostral regions where the lateral
edges of the sling are present in
Nfia+/ mice
(Fig. 4C, white arrow), the sling cells are
generated in Nfia/
mice (Fig. 4D, white arrow) but also
migrate into the septum at this level (Fig. 4D,
black arrow).
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Figure 4.
Abnormal development of the glial sling in the
Nfia/
mice. Expression of NeuN immunohistochemistry at E17 in either
Nfia+/
(A, C) or
Nfia/
(B, D) mice is shown. In heterozygote
mice, the glial sling forms a continuous band of cells across the
midline that spans the two cerebral hemispheres (A,
arrow). In
Nfia/
mice, sling cells migrate aberrantly into the septum (B,
arrow). In more rostral regions, the lateral edges of
the sling are present in the heterozygote (C,
arrow) and the knock-out (D, white
arrow), but at this level sling cells also migrate
aberrantly into the septum in the knock-out (D,
black arrow). Scale bar: (in D)
A-D, 150 µm.
|
|
Abnormal development of midline commissural projections: the corpus
callosum and the hippocampal commissure
We examined the development of medial cortical projections in the
Nfia knock-out mice to investigate when the callosal
abnormality is first evident. We used DiI labeling between E15 and P0
to view the trajectory of axons during the period in which the corpus callosum normally forms in mice. At E15 we observed no difference in
the cortical projections among Nfia+/+,
+/, or
/
littermates (data not shown). This is just before the time when the
first axons from the cingulate cortex cross the midline to pioneer the
corpus callosum in mice (Rash and Richards, 2001). At E17, however,
when a significant number of axons have crossed the midline in the
Nfia+/+ or
Nfia+/ mice
(n = 7/7) (Fig.
5A,C,E),
fewer axons appeared to reach the midline in the
Nfia/
mice (n = 6/6) (Fig. 5B), and those that
had, failed to cross (Fig.
5D,F). In both the genu
(Fig. 5, compare C, D) and the body (Fig. 5,
compare E, F) of the corpus callosum (or
where it would normally form), cortical axons in the
Nfia/
mice reached the midline and instead of turning to cross the midline,
grew down into the septum, ignoring the corticoseptal boundary. Once in
the septum, some axons began to turn laterally, forming the
characteristic beginnings of Probst bundles (Fig. 5F,
arrow). At E18 when significantly more axons have normally crossed the midline (Fig. 5G), swirls of axons
characteristic of Probst bundles were present on either side of the
midline in the
Nfia/
mice (Fig. 5H, arrow).
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Figure 5.
Development of callosal projections in
Nfia/
mice. Fixed brains at either E17 (A-F) or
E18 (G, H) were injected with DiI to label
callosal projections. In heterozygote mice, callosal axons crossed the
midline at E17 and E18 (A, C, E, G) but failed to
cross the midline in the knock-out (B, D, F, H).
Instead, these axons projected aberrantly into the septum and began to
curl back on themselves, characteristic of the formation of Probst
bundles (D, F, H, arrows). Scale bar: (in
H) A, B, 600 µm;
C, E, 200 µm; D,
F, 100 µm; G, H, 150 µm.
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|
In the most caudal regions of the corpus callosum (the splenium),
callosal axons (Fig. 6, red
axons) cross the midline dorsal to the hippocampal commissure
(Fig. 6, green axons). The hippocampal commissure forms over
an extended period of development from E14.8 to birth (Wahlsten, 1981;
Valentino and Jones 1982; Super and Soriano, 1994; Livy and Wahlsten,
1997). By E17, many axons of the hippocampal commissure have crossed
the midline in Nfia+/+ or
Nfia+/ mice
(n = 3/3) (Fig. 6C). In the
Nfia/
mutant, we did not observe any axons crossing the midline to form the
hippocampal commissure at E17 (n = 3/3) (Fig.
6B,D). Furthermore, the two
commissures normally remain distinct at this age, with the callosal
axons crossing dorsal to the hippocampal commissure (n = 3/3) (Fig. 6A,C). In the
Nfia/
mice, as in the body and the genu, callosal axons of the splenium did
not cross the midline at E17. Within the rostral (Fig.
6B) and caudal (Fig. 6D) regions of
the splenium at E17, callosal axons (red) and axons of the
fornix (Fig. 6B, green) or the hippocampal commissure (Fig. 6D, green) overlapped at
the border of the two commissures and did not remain in distinct axonal
bundles (n = 2/2) (Fig.
6B,D, arrows). At P0 we
also observed aberrations in both the hippocampal commissure and the
corpus callosum. As shown in the Nfia+/+
or Nfia+/
controls, the callosal axons (red) cross the midline in a
distinct bundle dorsal to the fornix (Fig. 6E,
green) and the hippocampal commissural axons (Fig.
6G, green) (n = 5/5); however, in
the Nfia/
mice, we again observed overlap of the axons in the corpus callosum and
fornix (Fig. 6F, arrow) and the corpus
callosum and the hippocampal commissure (n = 4/4) (Fig.
6H, arrow). In addition, at P0 we did observe some cases (n = 2/4) in which a few axons from
the hippocampus and occasionally a small number of callosal axons that
appeared to leave the Probst bundle crossed the midline together (Fig. 6F, arrowhead). This occurred only in the
splenium but not in the body and genu regions of the callosum at P0
(n = 4/4).
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Figure 6.
Development of the caudal corpus callosum and the
hippocampal commissure in
Nfia/
mice. Fixed brains at either E17 (A-D) or
P0 (E-H) were injected with both DiI to
label the corpus callosum (red axons) and DiA to label
the fornix and the hippocampal commissure (green
axons). The caudal regions of the corpus callosum form a
separate axon tract overlying the more ventrally located hippocampal
commissure. The hippocampal commissure is shown crossing the midline
(C) in the heterozygote; the fornix is present
underlying the caudal corpus callosum in a slightly more rostral
section (A). At E17 in the knock-out, axons of
both the corpus callosum and the hippocampal commissure fail to cross
the midline at both rostral and caudal levels. Instead, the axons of
the two commissures aberrantly mix together (B,
D, arrows). At P0, both commissures
remain as separate bundles in
Nfia+/
mice in both rostral (fornix region) (E) and
caudal regions where the hippocampal commissure is clearly evident
(G). In some knock-outs the mixed axon bundle
(F, arrow), containing both hippocampal
and callosal axons, crosses the midline (F,
arrowhead). In other areas the callosal axons do not
cross (H, arrow), but the hippocampal
commissure crosses alone (H, arrowhead).
Scale bar: (in H) A-D,
300 µm; E, G, 100 µm;
F, H, 200 µm.
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|
All results presented are from Nfia mice on a C57BL/6
background in which the
Nfia/
mice die at birth (das Neves et al., 1999). The variability in the
hippocampal commissure phenotype on the C57BL/6 background prompted us
to review our previous data from adult
Nfia/
mice on a mixed (129/C57BL/6/Swiss) background, of which ~5% survive
to adulthood. We found that the corpus callosum was absent in four of
four
Nfia/
mice on the mixed background and that the hippocampal commissure was
absent in three of four animals and severely reduced in one of four
animals (R. M. Gronostajski, unpublished observation). We did not
determine, in the few cases in which a hippocampal commissure did form
in adults, whether there were callosal axons growing within this bundle.
Slit2 and Robo1 and 2 expression in
Nfia/
mice
Our previous results showed that Slit2, expressed by the glial
wedge, could act as a chemorepellent molecule for callosal axons (Shu
and Richards, 2001). Furthermore, in
Slit2/
mice, callosal axons dive into the septum and ignore the glial wedge
(Bagri et al., 2002). Given that the phenotype of the
Nfia/
mice in some ways resembled that of the
Slit2/
mice and that glial wedge formation was disrupted in these mice, we
examined whether the phenotype may be caused by alterations in the
expression of Slit2 or Robo1 or Robo2 in these mice. We performed both
real time quantitative PCR on whole brains and in situ
hybridization using probes against Slit2, Robo1, or Robo2. Using real
time quantitative PCR we could detect no discernable difference in
expression among Nfia +/+,
+/, and
/
mice (data not shown). By in situ hybridization, however, we found a slight decrease in Slit2 expression at the midline in the
region of the glial wedge and a loss of expression in the indusium
griseum (Fig. 7, compare A,
C with B, D). Robo1 and Robo2 expression appeared unchanged in the cortical plate (Fig. 7, compare E, F for Robo1, and G, H
for Robo2).
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Figure 7.
Slit and Robo expression in the
Nfia mutant. E17
Nfia/
brains (B, D, F,
H) or brains from wild-type littermates
(A, C, E, G) were processed for in
situ hybridization using S35-labeled probes
of Slit2 (A-D), Robo1 (E,
F), and Robo2 (G, H). Slit 2 was
still expressed in the mutant, but expression in the indusium griseum
was absent (compare A, B, region marked
by arrows). At higher power it was also evident that
Slit2 expression in the glial wedge was decreased (compare
C and D, which correspond to the
boxed region in A and B,
respectively). However, neither Robo1 nor Robo 2 expression in the
cortical plate was decreased (compare E with
F and G with H,
respectively; low-power views of each section are shown in the
insets). Scale bars: (in D)
A, B, 800 µm; C,
D, 150 µm; (in H):
E-H, 400 µm.
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|
The anterior commissure and the perforating pathway are present in
Nfia/
mice
Neurofilament immunohistochemistry allowed us to observe a number
of midline axonal pathways in the same sections. We found that the
perforating pathway (Hankin and Silver, 1988; Shu et al., 2001)
appeared relatively normal in the
Nfia/
mice. The perforating pathway is primarily derived from neurons in the
medial septum and the diagonal band of Broca but has a small reciprocal
projection from the ventral cingulate cortex (Shu et al., 2001). The
axons form a fasciculated bundle on either side of the midline and
project ipsilaterally (Fig.
8A,E,
arrowhead). When the perforating axons reach the
corticoseptal boundary, they leave the midline, turning laterally and
then medially, avoiding the glial wedge. They perforate the corpus
callosum and project across it to the ventral cingulate cortex (Figs.
8B,F, arrow). A
precallosal component of the perforating pathway projects in a similar
manner but rostral to the genu of the corpus callosum (Fig.
8A,E, arrow). Although
the corpus callosum did not form, the perforating pathway still crossed
over the tangle of callosal axons on either side of the midline (Fig.
8F, arrow). The perforating axons did
project more laterally than in control mice before turning medially,
probably because of the malformation of the glial wedge. As noted
previously, at E17 the hippocampal commissure was not present in the
Nfia/
mice, and this was also evident by neurofilament labeling (Fig. 8,
compare D, H, arrowheads). In
addition, the fornix was greatly reduced in size in the
Nfia/
mice (Fig. 8G, arrow) compared with the
heterozygote littermate (Fig. 8C, arrow). The
anterior commissure (Fig. 8D,H,
arrow) still crossed the midline in the
Nfia/
mice, despite the fragility of this region caused by the absence of the other midline commissures. As previous analysis showed with GFAP labeling, however, the glial tunnel was greatly reduced surrounding a smaller anterior commissure, particularly in the rostral-caudal dimension (Fig. 2H,
arrow).
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Figure 8.
The perforating pathway and the
anterior commissure form normally in
Nfia/
mice. E17 brains of either
Nfia+/
(A-D) or
Nfia/
(E-H) mice were sectioned and processed for
neurofilament immunohistochemistry. Sections shown are in rostral
(A, E) to caudal (D, H) order. In
rostral regions, axons of the perforating pathway project along the
midline (A, E, arrowheads)
and then deviate more laterally at the corticoseptal boundary
(A, E, arrows). In more
caudal regions, the perforating axons cross over and "perforate"
the corpus callosum (B, arrow). Even in
the knock-out, perforating axons grow around the axon bundles on either
side of the midline without significantly deviating from their normal
trajectory (F, arrow). In more caudal
regions the fornix is evident in the heterozygote (C,
arrow) but reduced in the knock-out (G,
arrow). Furthermore, the corpus callosum and the
hippocampal commissure fail to cross (G,
H, arrowheads; arrowhead in
D shows the corpus callosum and the hippocampal
commissure crossing in caudal regions of the heterozygote) at E17, as
seen with carbocyanine dye labeling (Fig.
6B,D). The anterior
commissure crosses the midline in both wild-type and
Nfia/
mice (arrows in D and H,
respectively), despite the large cleft formed by the absence of the
corpus callosum and the hippocampal commissure in the knock-out. Scale
bar: (in H) A-H,
300 µm.
|
|
| |
Discussion |
We have shown that midline glial populations, the glial wedge, and
the indusium griseum glia, fail to develop normally in Nfia/
mice. In addition to the aberrant development of these glial structures, sling cells migrate abnormally into the septum. Together these structures make up the corticoseptal boundary that normally prevents callosal axons from entering the septum. Therefore, in Nfia/
mice, callosal axons fail to cross the midline and in some cases project ventrally into the septum. Furthermore, in more caudal regions,
callosal axons and axons of the hippocampal commissure cross within the
same region. These data indicate that defects in the formation of
midline structures are correlated with defects in midline commissure formation.
In Nfia knock-out mice, GFAP expression is greatly reduced
(das Neves et al., 1999). However, Nfia knock-outs display
no change in the expression of other glial markers including
proteolipid protein (das Neves et al., 1999) and BLBP (see Results).
NFI can bind to the GFAP promoter and regulate GFAP expression in
vitro (Miura et al., 1990; Krohn et al., 1999) and therefore may
play a similar role in vivo. Because GFAP is a major
structural component of glial processes, a decrease in GFAP may affect
the extension of glial processes in
Nfia/
mice. The few glial wedge processes that remain in the knock-out appeared shorter, indicating that they may be unable to adequately extend their processes. BLBP labeling confirmed a reduction or complete
absence of the formation of glial processes in the glial wedge and
indusium griseum glia. A reduction in GFAP by itself is unlikely to be
the cause of ACC in the
Nfia/
mutant. Although a reduced size of the corpus callosum attributable to
aberrant myelination has been noted in one GFAP knock-out mouse (Liedtke et al., 1996), no callosal changes have been detected in two
other GFAP knock-out strains (McCall et al., 1996; Shibuki et al.,
1996).
Although
Nfia/
mice display a reduction in midline glial processes, their cell bodies
may still be present or they may also be reduced. Slit2 is expressed by
these midline glial populations. Therefore, a reduction in the
expression of Slit2 at the corticoseptal boundary by in situ
hybridization may be caused by a reduction in the number of glia within
this region. Nfia is not essential for Slit2 expression
because we could detect no overall decrease in Slit2 expression by real
time PCR between wild-type and Nfia knock-out animals (data
not shown). The reduction in Slit2 RNA levels also suggests that
reduced levels of Slit2 protein may be presented to the callosal axons.
We proposed previously that Slit2 expressed by glia within the indusium
griseum above the corpus callosum, and by the glial wedge below the
corpus callosum, may provide a surround repulsion mechanism that keeps
callosal axons confined to the correct path and repels them toward and across the midline (Shu and Richards, 2001). Because no Slit2 seems to
be expressed in the indusium griseum, this surround repulsion mechanism, if it exists, would be lost. These data suggest that the
acallosal defect in the Nfia mutant could be caused by
defects in the development of midline glia and a reduction in Slit2
expression at the midline.
The formation of the midline zipper glial population (Silver et al.,
1993) was also disrupted in the
Nfia/
mice. These glia were either absent or their position shifted in
Nfia/
mice. Midline zipper glia are proposed to be essential for midline fusion at the corticoseptal boundary (Silver et al., 1993), and therefore a disruption in their development could cause abnormalities in midline fusion and consequently callosal formation. Although we did
not observe gross defects in midline fusion by light microscopic analysis,
Nfia/
mice did display small regions along the dorsoventral midline where
"spot" adhesions rather than complete fusion may have occurred. Further analysis at the electron microscopic level is required to
determine the full extent of midline fusion defects in
Nfia/
mice. Furthermore, because very little is known about the molecular mechanisms of how midline fusion actually occurs, it remains to be
investigated whether Nfia is directly involved in mediating such processes.
Although it is possible that loss of Nfia results in an
intrinsic axonal defect, the findings that Nfia is not
expressed in most callosal neurons early in callosal formation and that
Robo1 and Robo2 expression are unaffected in Nfia knock-out
mice (Fig. 7) makes this less likely. However, further experiments are
required to rule out an axon intrinsic defect in the Nfia mutant.
NFI-A is highly expressed in the glial sling, and sling cell migration
was greatly disrupted in the knock-out. If sling cells migrate along
the glial wedge to the midline, this migration defect could be an
indirect effect of the glial wedge malformation. Alternatively, Nfia itself may regulate either the migration or
proliferation of sling cells. If the lack of Nfia caused the
sling cells to continue to proliferate, then their proliferative state
may disrupt their ability to follow and respond to environmental cues
as they migrate. Previous studies have shown that in addition to cues within the cellular environment, a critical determinant for
layer-specific cell migration in the cerebral cortex is whether a cell
has completed S phase of the cell cycle (McConnell and Kaznowski, 1991;
Frantz and McConnell, 1996; Desai and McConnell, 2000). Analysis of
proliferation and apoptosis rates in the region encompassing the
corticoseptal boundary should allow us to distinguish between these hypotheses.
We also observed that the hippocampal commissure failed to form in all
embryonic and most of the postnatal mutants. However, this phenotype
was variable between animals, indicating that compensatory mechanisms
such as the expression of other NFI family members may be involved. In
the most caudal regions of the corpus callosum, we found that callosal
axons overlapped with axons of the fornix and hippocampal commissure
axons, particularly at the border between the two commissures. This
overlap of callosal and hippocampal axons could occur either because of
defects in glial wedge and sling development, which may help to
separate the two commissures, or because Nfia regulates the
expression of surface molecules on the hippocampal axons themselves
that normally keeps them separated from the callosal axons. We also
observed a marked reduction in the size of the fornix and the
hippocampal commissure and a reduction in GFAP immunohistochemistry
within the fimbria region in the Nfia/
mice. NFI-A is expressed throughout the hippocampus, particularly within the dentate gyrus (Plachez et al., 2001), and therefore may be
involved in the development of both neurons and glia within the hippocampus.
In addition to the corpus callosum and the hippocampal commissure
defects, we observed a defect in the formation of the glial tunnel and
the anterior commissure. Although some axons crossed the midline, even
in the absence of other midline commissures, the rostrocaudal size of
the anterior commissure was greatly reduced. Again, this may reflect
either a role for Nfia in the development of neurons in the
olfactory bulb that express Nfia (Plachez et al., 2001) and
project through the anterior commissure or an indirect effect via the
glial tunnel (Katz et al., 1983; Silver et al., 1993; Cummings et al.,
1997; Pires-Neto et al., 1998), which was also malformed. At present it
is not known whether the glial tunnel expresses guidance molecules for
axons of the anterior commissure. It is possible that similar
mechanisms operate in the development of the corpus callosum and the
formation of other midline commissures.
The perforating pathway formed normally in the Nfia mutant,
although it crossed over the Probst-like bundles to enter the cingulate
cortex. NFI-A is not expressed in the medial septum/diagonal band of
Broca region where most of the neurons that give rise to the
perforating pathway reside (Shu et al., 2001). Perforating axons avoid
the glial wedge morphologically in wild-type animals (Shu et al.,
2001), but our data in the Nfia mutant suggest that the
formation of the glial wedge is not required for the projection of the
perforating axons into the cingulate cortex.
Our results indicate that Nfia may play a critical role in
regulating genes involved in glial and/or neuronal development and
axonal guidance. Given the overlapping expression of Nfib and Nfix in the brain, it is interesting that the mutation
of Nfia had such profound effects on these developmental
processes. Perhaps this indicates that different family members perform
different functions within the same cells and may not provide
compensation for each other. Indeed, major differences in
transcriptional modulation properties have been seen between different
NFI gene products in transient transfection assays (Apt et al., 1994;
Chaudhry et al., 1998) (see latter for additional references). Given
the complexity of alternatively spliced transcripts of these genes, it
will be important to analyze the expression of specific protein
isoforms of each NFI gene. It will also be important to identify
downstream targets of Nfia to determine at a molecular level
how Nfia is involved in the development of glia and neurons
and the formation of midline commissures.
| |
FOOTNOTES |
Received Feb. 11, 2002; revised Oct. 8, 2002; accepted Oct. 14, 2002.
This work was supported by March of Dimes Foundation for Birth Defects
Grant 5-FY99-842 (L.J.R.) and by National Institutes of Health Grants
NS37792 (L.J.R.) and HD34908 (R.M.G.). We thank Kimberley M. Valentino
for excellent technical assistance.
Correspondence should be addressed to Dr. Linda J. Richards, Department
of Anatomy and Neurobiology, HSF 222, The University of Maryland,
Baltimore, 685 West Baltimore Street, Baltimore, MD 21201. Email:
lrich001{at}umaryland.edu.
K. G. Butz's and R. M. Gronostajski's present address:
Department of Biochemistry, State University of New York at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, NY 14214.
| |
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TOP
ABSTRACT
Introduction
