Genes and Development Research Group, Hotchkiss Brain Institute, University of Calgary Faculty of Medicine, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
* Author for correspondence (e-mail: weiss{at}ucalgary.ca)
Accepted 23 November 2004
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SUMMARY |
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Key words: Ventricular zone, Subventricular zone, Self-renewal, Ciliary neurotrophic factor, Leukemia inhibitory factor, Mouse
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Introduction |
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VZ precursors of the LGE express the homeobox transcription factor GSH2
(Hsieh-Li et al., 1995;
Yun et al., 2003
), while more
committed precursors in the SVZ are distinguished by increased expression of
the proneural basic helix-loop-helix transcription factor, MASH1 (Ascl1 -
Mouse Genome Informatics) (Casarosa et al.,
1999
; Fode et al.,
2000
; Porteus et al.,
1994
; Torii et al.,
1999
) and the Distal-less related homeobox genes, DLX1 and DLX2
(Anderson et al., 1997
;
Eisenstat et al., 1999
;
Porteus et al., 1994
;
Yun et al., 2002
). It has been
suggested that MASH1 modulates Notch signaling in the germinal zone by
regulating expression of the Notch ligands Delta1/3, and in the
absence MASH1 (and hence Notch signaling) VZ precursors take on SVZ
characteristics (Casarosa et al.,
1999
; Yun et al.,
2002
). Furthermore, overexpression of a proneural gene
functionally related to MASH1, neurogenin 2 (NGN2), has been demonstrated to
promote the acquisition of a SVZ fate by cells born in the cortical VZ
(Miyata et al., 2004
). These
studies strongly suggest major roles for Notch signaling and proneural gene
activity in the establishment and/or maintenance of the VZ and SVZ germinal
compartments in the telencephalon.
We have previously reported that ciliary neurotrophic factor (CNTF)
promotes the self-renewal of embryonic forebrain epidermal growth factor
(EGF)-responsive neural stem cells (NSCs) in vitro
(Shimazaki et al., 2001) by
increasing the expression of NOTCH1 and inhibiting the expression of MASH1
(Chojnacki et al., 2003
),
potentially conferring a more VZ like phenotype. CNTF signaling is transduced
by a heterotrimer composed of the CNTFR
/LIFRß/gp130 receptor
complex, whereas LIF signaling is mediated by the LIFRß/gp130
heterodimer, both of which can activate the JAK-STAT signaling cascade
(Taga and Kishimoto, 1997
).
Others have found that LIF and gp130-mediated signaling can regulate the
self-renewal of cortical NSCs in vivo
(Hatta et al., 2002
). In the
present study, we tested the hypothesis that CNTF/LIF/gp130 receptor signaling
contributes to the formation and maintenance of the germinal layers in the LGE
during early stages of development. Our results suggest CNTF/LIF/gp130
receptor signaling is both necessary and sufficient to promote the
self-renewal/expansion of a subpopulation of VZ precursors, establishing a VZ
precursor differentiation gradient that is required for the normal growth of
the ventral forebrain.
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Materials and methods |
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Neural stem cell culture and growth factors
The generation and differentiation of neurospheres derived from the E14 CNS
was performed as previously described with minor modifications
(Represa et al., 2001;
Reynolds et al., 1992
;
Shimazaki et al., 2001
).
Briefly, the lateral and medial ganglionic eminences or cervical/thoracic
regions of the spinal cord were dissected from E14 mouse embryos, mechanically
dissociated and plated as primary cultures at a density of 100,000 cells/ml in
the presence of FGF2 (20 ng/ml; R&D Systems, Minneapolis, MN) and heparin
sulfate (2 µg/ml; Sigma, St Louis, MO). After 7 days in vitro (DIV), the
resulting primary neurospheres were mechanically dissociated and plated as
pass 1 cells at 50,000 cells/ml. Additional cytokines used were CNTF (20
ng/ml; a generous gift from Dr Robert Dunn, Montreal General Hospital Research
Institute) and LIF (20 ng/ml; Chemicon, CA, USA). To dissociate pass 1
neurospheres, we used a 3-minute treatment of 3 ml trypsin-EDTA (Invitrogen,
Canada) at 37°C, followed by 5 ml trypsin inhibitor (Sigma). Cells were
centrifuged at 600 rpm for 10 minutes and resuspended in 5 ml of trypsin
inhibitor and mechanically dissociated with a fire-polished pipette. Cells
were then suspended in 8 ml of basal media, centrifuged, resuspended in 2 ml
of basal media and then used for experiments. For quantification of nestin+
cells, pass 1 neurospheres were dissociated, plated at 200,000 cells/ml on
poly-l-ornithine coated coverslips and fixed after 30 minutes for processing
for immunocytochemistry. For quantification of ß-tubulin III+ cells, pass
1 neurospheres were dissociated, plated at 200,000 cells/ml for 5 DIV and
fixed for immunocytochemical analysis. For quantification of NSC self-renewal,
dissociated pass 1 neurospheres were plated in 96-well plates at a density of
10,000 cells/ml and the number of secondary neurospheres was quantified after
10 DIV.
Area measurements of the LGE and cortex
The forebrains of E12.5 Lifr-/-,
Lifr+/- and Lifr+/+ embryos were serially
sectioned and collected onto two rounds of six slides with eight sections per
slide and the slides were stained with Hoechst (Sigma). Slides were randomly
sampled from each embryo and sections of the LGE and cortex were photographed
beginning at the most rostral appearance of the LGE and extending caudally to
the first appearance of the dorsal thalamus. The LGE was outlined and the area
was calculated using Image J software (NIMH, MD, USA). The areas of the
Lifr-/- LGE and cortex were normalized to the averaged
areas of Lifr+/- and Lifr+/+
littermates for statistical analysis.
Bromodeoxyuridine labeling, detection and quantification
Pregnant dams received a single injection of bromodeoxyuridine on E12.5
(BrdU; 120 mg/kg, ip; dissolved in 0.007% NaOH in phosphate buffer; Sigma) and
were sacrificed thirty minutes post-injection. The brain and upper thoracic
spinal cord was removed from E12.5 embryos and processed for
immunohistochemistry as described below. Brains and spinal cords were
sectioned serially onto two rounds of six slides with eight 14 µm sections
per slide. Sections were pretreated with 1 M HCl for 30 minutes at 60°C to
denature the DNA. Rat monoclonal anti-BrdU (1:50; Sera-Lab, Sussex, UK) and
biotinylated donkey anti-rat (1:200; Jackson ImmunoResearch, West Grove, PA)
with Streptavidin-Cy3 (1:2000; Jackson ImmunoResearch) were used for BrdU
detection. The number of BrdU-expressing cells in the LGE or cortex was
quantified on randomly sampled slides, beginning at the first appearance of
the dorsal thalamus and extending rostrally for five sections for each animal.
The same methodology was used for GSH2, MASH1 and pHH3 quantification.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)
Apoptosis was detected in tissue sections using the In Situ Cell Death
Labeling Kit (Roche; Indianapolis, IN) according to the manufacturer's
instructions with the following modifications. Sections were incubated in 100
µl Proteinase K (diluted 1:1000 from a stock solution of 20 µg/µl in
10 mM Tris-HCl pH 8.0) for 6 minutes at room temperature and then washed
extensively in PBS. The slides were incubated for 2 hours at 37°C in the
presence of the TUNEL reaction mixture supplied by the kit, were washed in
PBS, and then incubated overnight in the presence of mouse anti-FITC (1:400;
Sigma). Detection was performed using biotinylated goat anti-mouse (1:200;
Jackson ImmunoResearch) and Streptavidin-FITC (1:1000; Jackson
ImmunoResearch). We quantified only those cells that were positive on the FITC
channel and therefore not autoflourescent. Quantification was carried out in
the same manner as for BrdU and the number of TUNEL-positive cells was
normalized to the calculated area of the LGE, as described above.
Explant cultures
Explant cultures were performed as described previously
(Kingsbury et al., 2003) with
modifications. Each cerebral hemisphere was dissected away from the developing
E14.5 CNS in PBS. Next, the choroid plexus and regions of the septum and
medial cortex were carefully dissected away to allow the culture media access
to the ganglionic eminences. One hemisphere from each brain was placed in
basal media (Opti-MEM I; Gibco, NY, USA) and the other was placed in basal
media containing 50 or 100 ng/ml CNTF (either concentration was effective) in
six-well plates (Nunc). The explants were placed on a rocker in a tissue
culture incubator at 37°C, 5% CO2 in air, for 18.5 hours, fixed
in 4% paraformaldehyde at 4°C for 2 hours and cryoprotected in 25% sucrose
overnight. Tissue was cryosectioned and processed for immunohistochemistry and
quantification as described above for embryonic tissue. Explants that appeared
damaged or unhealthy were excluded from analysis.
RT-PCR analysis
The choroid plexus was dissected from the lateral ventricles of 10-12 E14.5
CD-1 embryos and pooled for each round of RNA extraction. RNA was isolated
using the RNeasy Protect Mini Kit (Qiagen, ON, Canada) and reverse transcribed
using the Superscript III First-Strand Synthesis Kit (Invitrogen, CA, USA)
with random hexamer primers, all according to the manufacturers instructions.
PCR analysis of the resulting cDNA was performed to determine the expression
of LIF using methods previously described
(Bauer et al., 2003). CNTF cDNA
was amplified by PCR for 35 cycles of 95°C for 45 seconds, 55°C for 45
seconds and 72°C for 45 seconds using 5'-ATGGCTTTCGCAGAGCAA-3'
as sense primer and 5'-CTACATTTGCTTGGCCC-3' as the antisense
primer (Subang et al., 1997
),
which amplified a 596 bp fragment. CNTF and LIF RT-PCR products were sequenced
by the University of Calgary DNA Sequencing Laboratory and checked for
accurate identification by a BLAST search of the mouse genome.
Antibodies and immunohistochemistry
The primary antibodies used in this study were as follows: rabbit
anti-NOTCH1, anti-FGFR1, anti-FGFR2, anti-LIFRß, anti-gp130, anti-CNTF
and anti-LIF (1:50, Santa Cruz Biotechnology); goat anti-CNTFR (1:50,
Santa Cruz Biotechnology); mouse anti-Ki67 (1:100; NovoCastra Laboratories,
Newcastle, UK); mouse anti-ß-III tubulin (1:100; Sigma); rabbit anti-GSH2
(generous gift from Dr Kenneth Campbell, Children's Hospital Research
Foundation, Cincinnati); mouse anti-MASH1 (generous gift from Dr David
Anderson, Caltech); rabbit anti-DLX1 and rabbit anti-DLX2 (1:100; generous
gifts from Dr David Eisenstat; University of Manitoba); mouse anti-GAD65
(1:100; BD Pharmingen); and rabbit anti-phospho Histone H3 (pHH3; 1:100;
Upstate Biotechnologies).
For immunohistochemical analysis on embryonic tissue sections, timed pregnant mice were killed, the brain or upper thoracic spinal cord was removed from the embryos, fixed in 4% paraformaldehyde for 2 hours at 4°C and then cryoprotected by 10% sucrose and then 25% sucrose solutions overnight. All tissue was embedded in Tissue Tek OCT compound (Sakura Finetek, Torrance, CA) and cryosectioned at 14 µm. For receptor staining, tissue was post-fixed in methanol at -20°C for 8 minutes. Primary antibodies were followed by incubation with fluorescein- or rhodamine-conjugated secondary antibodies against mouse, rabbit or goat IgG, or by using biotin-conjugated secondary antibodies (all 1:200) followed by Streptavidin-CY3 (1:2000; Jackson ImmunoResearch). For nuclear counterstaining, Hoechst was used (1:100; Sigma).
Statistical analysis
Values are mean±s.e.m. Statistical significance between groups was
assessed using ANOVA and the Tukey Honest Significant Difference Test, or
paired t-test where noted.
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Results |
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The relationship between CNTF/LIF/gp130-mediated signaling and FGF signaling with respect to NSC regulation is not known. To assess whether CNTF or LIF signaling modulates FGF-responsive NSC proliferation, NSCs residing within the E14.5 ganglionic eminences were isolated and grown as neurospheres in FGF2. These neurospheres were dissociated, reseeded at 50,000 cells/ml in FGF2 and the presence or absence of either CNTF or LIF, and the total number of cells generated in each growth condition was assessed after 7 DIV. Remarkably, the number of cells generated by ventral forebrain NSCs in the presence of FGF-2 (Fig. 1M; 2.9±0.3x106) doubled in response to the addition of either CNTF (6.0±0.8x106; P=0.009; n=5) or LIF (5.4±0.5x106; P=0.03; n=5). To determine whether or not this increase might be due to an increase in the total number of neurospheres generated, primary FGF2 neurospheres were dissociated and replated in 96-well plates in FGF2 and the presence or absence of CNTF or LIF. After 10 DIV, we found no difference in the number of neurospheres generated in the presence of CNTF or LIF relative to FGF2 alone (Fig. 1N); however, the size of the neurospheres increased in CNTF- and LIF-treated conditions (Fig. 1O). Quantification revealed over a two-fold increase in the number of large neurospheres (>200 µm in diameter) in the CNTF and LIF growth conditions compared with FGF2 alone (Fig. 1P; CNTF, P=0.0006; LIF, P=0.005; n=3).
One possible explanation for the increased size of the CNTF or LIF treated
neurospheres is that CNTF/LIF/gp130 signaling promotes NSC self-renewal and
expansion, as we suggested previously for EGF-responsive NSCs
(Shimazaki et al., 2001). To
determine the number of precursors within the neurospheres cultured in the
absence or presence of CNTF or LIF, we counted (in each growth condition) the
percentage of cells that expressed nestin, a marker of undifferentiated cells
(Frederiksen and McKay, 1988
).
In the CNTF/LIF-treated cultures, we observed a
50% increase in the
percentage of cells expressing nestin relative to cultures generated in FGF2
alone (Fig. 1Q; CNTF
P=0.03; LIF P=0.04; n=3). Additionally, when
neurospheres generated in either FGF2, FGF2+CNTF or FGF2+LIF were dissociated
and replated in 96-well plates in the presence of FGF2, the number of
secondary neurospheres generated after 10 DIV doubled in the CNTF- and
LIF-treated cultures (Fig. 1R;
CNTF, P=0.02; LIF, P=0.02; n=3). These results
suggest that CNTF/LIF/gp130 signaling promotes the self-renewal/expansion of
FGF-responsive NSCs residing within the VZ.
CNTF/LIF/gp130 signaling does not promote the self-renewal/expansion of spinal cord NSCs
Unlike the forebrain, the developing spinal cord has only a single germinal
layer, called the VZ. We therefore sought to determine whether CNTF/LIF/gp130
signaling might play a different role in the regulation of spinal cord NSCs
compared with forebrain NSCs. At E11.5, FGFR2 expression is clearly present in
the spinal cord VZ (Fig. 2A).
However, CNTFR expression is primarily in the mantle zone
(Fig. 2B), the region of
neuronal maturation as shown by ß-tubulin III expression
(Fig. 2C). By E14.5, the spinal
cord germinal zone is reduced in size, as shown by FGFR2 expression
(Fig. 2D). The expression of
CNTFR
(Fig. 2E)
continued to predominate in the mantle zone
(Fig. 2F), although low levels
of expression are evident in the VZ. These results suggest that
CNTF/LIF/gp130-mediated signaling might primarily influence cells of the
neuronal lineage in the mantle zone rather than NSCs within the VZ of the
spinal cord.
|
Previous work has demonstrated a role for CNTFR and LIFRß
signaling in regulating the differentiation/survival of spinal motoneurons
(Li et al., 1995
;
Murphy et al., 1997
;
Nakashima et al., 1999
;
Richards et al., 1992
). Given
that we observed the majority of CNTFR
expression in the mantle zone,
we asked whether the addition of CNTF/LIF to spinal cord neurosphere cultures
affected neurogenesis by quantifying the number of ß-tubulin III+
neurons. The addition of either CNTF (**P=0.007;
n=4) or LIF (**P=0.0001; n=4) to E14.5
pass 1 spinal cord neurosphere cultures resulted in a doubling of the number
of neurons generated by spinal cord NSCs, compared with FGF2 alone
(Fig. 2H). By contrast, there
was no change in the number of neurons generated by E14.5 ventral forebrain
neurosphere cultures treated with either CNTF or LIF
(Fig. 2I; n=3).
CNTF/LIF/gp130 signaling is required in vivo for normal growth of the developing forebrain
To determine whether CNTF/LIF/gp130 signaling was required in vivo for the
normal growth of the forebrain during development, we analyzed Lifr
null mutant mice, in which both CNTF and LIF signaling are disrupted. The
forebrains of E12.5 Lifr+/+, Lifr+/-
and Lifr-/- littermate embryos were serially sectioned and
the gross morphologies of the forebrains were compared. No obvious difference
in forebrain growth was observed between the Lifr+/+ and
Lifr+/- littermates (data not shown). On the other hand,
although an obvious difference in overall brain size was not noted, a
histological comparison of sections counterstained with the nuclear marker
Hoechst from rostral to caudal between
Lifr+/+/Lifr+/- and
Lifr-/- mice revealed gross morphological differences that
were suggestive of impaired growth in the mutant forebrains
(Fig. 3). At rostral levels,
the most obvious difference was a decrease in the size of the LGE in the
Lifr-/- mice (Fig.
3A,B). At more caudal regions of the forebrain, we observed that
the cortex appeared thinner compared with
Lifr+/+/Lifr+/- controls, and the LGE,
MGE and caudal ganglionic eminence were also reduced in size
(Fig. 3C-H). This phenotype was
partially penetrant with 56% (n=9) of the Lifr-/-
embryos displaying gross morphological changes relative to littermate
Lifr+/+/Lifr+/- controls.
|
GSH2, MASH1 and DLX2 expression define a gradient of VZ precursor differentiation in the LGE
To investigate the role of CNTF/LIF/gp130 signaling in the formation and
maintenance of the ventral forebrain germinal layers, it was necessary to
establish criteria to distinguish undifferentiated VZ precursor cells from
more differentiated precursors. Previous studies have suggested that the
transcription factors GSH2, MASH1 and DLX2 might be useful as markers of VZ
precursor differentiation (Eisenstat et
al., 1999; Porteus et al.,
1994
; Yun et al.,
2002
; Yun et al.,
2003
), prompting us to examine their expression in more
detail.
At E8.5, neither GSH2 nor MASH1 were expressed in the VZ (data not shown),
in agreement with previous studies (Yun et
al., 2002), and we have designated these cells rudimentary
precursor cells (RPs). At E9.5, when the germinal zone is composed of a VZ
only, a large number of precursor cells expressed GSH2 in the LGE, and we have
designated these as P1 precursor cells
(Fig. 4A). A subpopulation of
VZ precursors also expressed MASH1 (Fig.
4B), and all the MASH1+ cells co-expressed GSH2
(Fig. 4C). Very few DLX2+ cells
were detected at the pial surface of the E9.5 LGE VZ
(Fig. 4D), and these cells
co-expressed MASH1 (Fig. 4E,F).
The DLX2+ subpopulation was presumed to express GSH2, as all MASH1+ cells at
this stage appeared to express GSH2 (the triple labeling experiment was not
possible). This analysis, at a stage when the SVZ is absent
(Smart, 1976
), suggests that
the LGE VZ is composed of P1, P2 and P3 precursors, as depicted in
Fig. 4P.
|
To understand how CNTF, LIF and FGF signaling might regulate this
differentiation gradient, we performed a series of double-labeling
experiments. Analysis of the expression of CNTFR and GSH2 revealed that
a subpopulation of GSH2+ VZ precursors located close to the ventricular
surface express CNTFR
(Fig.
4M; arrow indicates a double-labeled cell). LIFRß, required
for both CNTF and LIF signaling, is also expressed by VZ precursors close to
the ventricular surface. Double labeling revealed that very few LIFRß+
cells co-express MASH1 (Fig.
4N; arrow indicates an example of a double-labeled cell).
Similarly, only a small subpopulation of MASH1+ cells co-expressed FGFR2
(Fig. 4O). These results
suggest that CNTF, LIF and FGF signaling might act primarily to regulate the
self-renewal/expansion of the VZ P1 precursor (GSH2+MASH1-) cell population,
perhaps inhibiting progression to more differentiated precursor fates (P2-P5),
as depicted in the schematic in Fig.
4P.
CNTF/LIF/gp130 signaling is required in vivo to maintain the VZ precursor differentiated gradient
We hypothesized that impaired self-renewal/expansion of P1 precursor cells,
owing to lost CNTF/LIF/gp130 signaling, results in the precocious
differentiation of VZ precursors. To test this, we analyzed the number of
GSH2+ VZ precursors in the LGE of E12.5 Lifr-/- animals
relative to Lifr+/+/Lifr+/- embryos.
Lifr-/- embryos had 30% fewer GSH2+ precursors
(Fig. 5B; 911±23)
relative to Lifr+/+/Lifr+/- controls
(Fig. 5A; 1324±44;
paired t-test **P=0.0006; n=4). When
normalized to the decreased area of the LGE, the overall decrease in the
Lifr-/- GSH2+ population was 15% relative to
Lifr+/+/Lifr+/- littermates (paired
t-test *P=0.04). By contrast, the number of
MASH1+ precursors within the germinal zone of the Lifr-/-
embryos (Fig. 5D;
802±30) was increased by 37% compared with
Lifr+/+/Lifr+/- embryos
(Fig. 5C; 504±22; paired
t-test **P=0.0007; n=3). Double-labeling
of MASH1 and GSH2 demonstrated a dramatic increase in the MASH1+GSH2-cell
population within VZ of the Lifr-/- embryos
(Fig. 5E,F). Analysis of DLX2
also revealed increased expression by VZ precursors within the
Lifr-/- LGE (Fig.
5G,H). Double labeling with MASH1 and DLX2 demonstrated that the
majority of the DLX2+ cells within the VZ of the Lifr-/-
embryos co-expressed MASH1 (Fig.
5I,J).
|
CNTF/LIF/gp130 signaling is sufficient to maintain a gradient of VZ precursor differentiation within the LGE
To understand better how CNTF/LIF/gp130 signaling might operate to maintain
VZ precursors, we attempted to determine the likely source of CNTF and LIF
acting upon the VZ during forebrain development. Immunostaining for CNTF
expression in the forebrain of E14.5 embryos revealed robust expression within
the choroid plexus, as well as at the base of the choroid plexus
(Fig. 6A,B; arrow and
arrowheads indicate staining). Immunoreactive cells were also observed within
the mantle zone of the LGE (Fig.
6B); however, it is unlikely that these cells would greatly
influence precursors within the VZ. LIF expression was also robust in the
choroid plexus (Fig. 6C,D). We
further confirmed the expression of both CNTF and LIF in the E14.5 choroid
plexus by RT-PCR (Fig. 6E). The
choroid plexus generates the cerebrospinal fluid (CSF) that bathes the
interior of the embryonic and adult brain, and it is known to secrete many
different growth factors and cytokines
(Chodobski and Szmydynger-Chodobska,
2001; Dziegielewska et al.,
2001
; Gard et al.,
2004
; Speake et al.,
2001
; Sturrock,
1979
). Our results suggest that the embryonic choroid plexus also
generates CNTF and LIF. The presence of these molecules in the CSF would place
them in an ideal position to influence precursors at the ventricular surface
and establish a gradient of VZ precursor differentiation, as depicted in the
model in Fig. 6F.
|
|
Previously, we have reported that CNTF signaling can regulate the
expression of NOTCH1 (Chojnacki et al.,
2003) and a disruption of Notch signaling has been suggested to
result in the precocious expression of SVZ characteristics by VZ precursors
(Casarosa et al., 1999
;
Yun et al., 2002
). Therefore,
we examined the expression of the NOTCH1 receptor in our explant culture
conditions. In the normal E14.5 LGE, NOTCH1 expression appeared in a gradient
opposite to MASH1, being highest at the ventricular surface and decreasing in
expression away from the surface (Fig.
7N). In the basal media explant condition NOTCH1 expression was
sustained; however, the gradient was disrupted and replaced by a cluster of
NOTCH1 immunoreactivity along the ventricular surface
(Fig. 7O; n=5). The
gradient was restored in the CNTF-treated explants
(Fig. 7P; n=5).
Double-labeling with NOTCH1 and MASH1 in the normal E14.5 LGE revealed that
MASH1 expression was restricted to cells expressing low levels of NOTCH1 at
superficial levels of the NOTCH1 gradient or by cells that did not express
NOTCH1 in the SVZ (Fig. 7Q).
Remarkably, in the basal media explants many of the clustered
NOTCH1-expressing cells co-expressed MASH1
(Fig. 7R; n=5), while
CNTF treatment maintained the graded separation of these two cell populations
(Fig. 7S; n=5).
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Discussion |
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CNTF/LIF/gp130 signaling maintains a gradient of VZ precursor differentiation
The signals that form and maintain the germinal layers of the forebrain are
poorly understood. Building on the work of Yun et al.
(Yun et al., 2002), we have
proposed that a gradient of VZ precursor differentiation can be defined by
GSH2, MASH1 and DLX1/2 expression. On this basis, precursor differentiation in
the LGE can be considered to occur in three major steps: (1) VZ precursors are
initially specified by expression of GSH1 and GSH2, which inhibit dorsal cell
fate characteristics by repressing Pax6
(Corbin et al., 2000
;
Schuurmans and Guillemot,
2002
; Toresson et al.,
2000
; Yun et al.,
2003
; Yun et al.,
2001
); (2) Precursors become initially committed to the neuronal
lineage by the expression of MASH1
(Casarosa et al., 1999
;
Fode et al., 2000
;
Nieto et al., 2001
;
Torii et al., 1999
;
Yun et al., 2002
); and (3)
MASH1 initiates the expression of the Dlx genes
(Fode et al., 2000
), which
induce glutamic acid decarboxylase expression and regulate the generation of
later born neuronal populations (Anderson
et al., 1997
; Eisenstat et
al., 1999
; Stuhmer et al.,
2002
; Yun et al.,
2002
). Recent time-lapse studies of cortical precursors have
demonstrated that the SVZ arises from asymmetric divisions of precursors in
the VZ (Miyata et al., 2004
;
Noctor et al., 2004
), and a
similar relationship probably exists between the VZ and SVZ in the LGE
(Halliday and Cepko, 1992
).
Therefore, the differentiation gradient we have described in the LGE
(summarized in Fig. 4P) may
represent differentiating daughter cells, asymmetrically born at the
ventricular surface from P1 precursors, trafficking through the VZ out to the
SVZ.
A role for Notch signaling and proneural gene expression in maintaining the
separate VZ and SVZ layers has been suggested. NGN2 promotes the commitment of
VZ daughter cells to the SVZ in the cortex
(Miyata et al., 2004). It is
likely that MASH1 plays a similar role in the ventral telencephalon, given its
known role in promoting precursor commitment and that NGN2 is sufficient to
rescue neurogenesis defects in the ventral telencephalon of MASH1 mutant mice
(Parras et al., 2002
). By
contrast, NOTCH1 signaling has been suggested to play a role in maintaining
the VZ population of the ventral forebrain
(Casarosa et al., 1999
;
Yun et al., 2002
). We have
previously demonstrated that CNTF/LIF/gp130 signaling supports embryonic NSC
self-renewal in vitro (Shimazaki et al.,
2001
) by promoting NOTCH1 and repressing MASH1 expression
(Chojnacki et al., 2003
). Our
present results suggest that FGF-responsive P1 precursors located at the
ventricular surface are the primary targets of CNTF/LIF/gp130 signaling in the
developing LGE. First, components of the CNTF/LIF/gp130 signaling pathway are
expressed primarily by GSH2+MASH1-precursors at the ventricular surface.
Second, this pathway promotes FGF-responsive NSC self-renewal/expansion in
vitro and promotes the P1 precursor fate in forebrain explant cultures.
Finally, in the absence of this signaling pathway VZ precursors precociously
differentiate and acquire characteristics of more differentiated cells at the
expense of the GSH2+ cell population. CNTF/LIF/gp130 signaling thus appears to
maintain an undifferentiated P1 precursor population in the VZ, which
contributes to the normal formation of two germinal layers having distinctly
different degrees of cellular commitment.
It is reasonable to suggest that CNTF/LIF/gp130 signaling may be acting
upstream of NOTCH1, as we have suggested previously
(Chojnacki et al., 2003), or in
coordination with NOTCH1 signaling
(Kamakura et al., 2004
) to
maintain the P1 precursor population. Our explant culture experiments reveal
that CNTF/LIF/gp130 signaling maintains NOTCH1- and MASH1-expressing cells
largely as separate populations. It may be that in the absence of this
signaling pathway fewer NOTCH1 receptors are expressed per P1 precursor cell,
resulting in decreased Notch signaling and the precocious expression of MASH1
and other more differentiated cellular characteristics. Alternatively, the
activation of STAT3 by CNTF/LIF/gp130 signaling may mediate important
crosstalk between the Notch and STAT signaling pathways [as recently suggested
by Kamakura et al. (Kamakura et al.,
2004
)], which is required to maintain the undifferentiated state
of P1 precursors.
A role for the choroid plexus in the maintenance of a stem cell niche in the developing forebrain
Our results suggest that the primary source of CNTF and LIF acting upon VZ
precursors may be the choroid plexus, a secretory structure that gives rise to
the CSF that bathes the interior of the developing brain
(Dziegielewska et al., 2001;
Speake et al., 2001
). The
choroid plexus is known to secrete many different growth factors and cytokines
into the CSF for delivery to the brain tissue
(Chodobski and Szmydynger-Chodobska,
2001
), appears as early as E11 in the forebrain
(Sturrock, 1979
), and is
believed to produce CSF throughout development
(Dziegielewska et al., 2001
).
The CSF of the developing embryonic forebrain is ideally positioned to
influence precursor cells within the germinal zone such that cells closest to
ventricular surface would be closest to the source of CNTF/LIF, while those
positioned farther away from this source would be in an environment
increasingly permissive for differentiation (see
Fig. 6F). In support of this
model, our results demonstrate that the removal of both the choroid plexus and
the CSF in the basal media explant condition results in the disruption of the
VZ precursor differentiation gradient. This defect was entirely rescued by the
addition of CNTF to the media bathing the explant and concurrently increased
the number of proliferating cells within the germinal zone.
It has been suggested that the adult choroid plexus does not express CNTF
and LIF (Gard et al., 2004),
which may be significant given that only the SVZ (and not the VZ) is
maintained into adulthood. Perhaps the production of CNTF and LIF by the
choroid plexus is required to maintain levels of these molecules sufficient
for the formation of both the VZ and SVZ germinal layers. This is an area for
future investigation that may be important for strategies aimed at the
intrinsic repair of the forebrain.
Distinct roles for CNTF/LIF/gp130 signaling in the ventral forebrain and spinal cord during development
Virtually all previously identified pathways that promote NSC
self-renewal/expansion are known to function in both the forebrain and spinal
cord. For example, FGF signaling promotes NSC self-renewal/expansion in both
the forebrain (Martens et al.,
2000; Raballo et al.,
2000
; Tropepe et al.,
1999
; Vaccarino et al.,
1999
) and spinal cord (Del
Corral and Storey, 2004
;
Represa et al., 2001
).
Similarly, Notch signaling promotes NSC self-renewal/expansion in both
forebrain (Chojnacki et al.,
2003
; Hitoshi et al.,
2002
; Schuurmans and
Guillemot, 2002
; Yun et al.,
2002
) and spinal cord regions
(Appel et al., 2001
;
le Roux et al., 2003
;
Lindsell et al., 1996
), as do
the membrane-associated proteins Numb and Numblike
(Petersen et al., 2002
;
Petersen et al., 2004
). Our
review of the literature suggests that a specific signaling pathway that
promotes VZ precursor self-renewal/expansion within the forebrain, but not the
spinal cord, has not been previously identified.
We found that the components of the CNTF/LIF/gp130 receptor complex were
robustly expressed by VZ precursors in the developing LGE, contrasting with
CNTFR and LIFRß expression in the spinal cord
(Fig. 2) (C.G. and S.W.,
unpublished), which was largely restricted to the mantle zone. A similar
pattern of CNTFR
expression was previously observed in the developing
rat spinal cord (MacLennan et al.,
1996
). This suggests that CNTF/LIF/gp130 signaling might have a
different role in the spinal cord compared with the forebrain. Indeed, CNTF
and LIF inhibited spinal cord NSC self-renewal/expansion in vitro, instead
promoting the generation/survival of neurons from FGF-responsive spinal cord
NSCs. Furthermore, we observed no impairment in the growth or number of
proliferating precursors in the developing spinal cord of
Lifr-/- mice and the addition of CNTF to spinal cord
explant cultures decreased the number of proliferating precursors in the
spinal cord VZ (see Fig. S3A,B in the supplementary material). Taken together,
these findings suggest that CNTF/LIF/gp130 receptor signaling specifically
contributes to growth of the forebrain (but not the spinal cord) during
development, by contributing to the formation and maintenance of the ventral
forebrain VZ.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/3/565/DC1
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ACKNOWLEDGMENTS |
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