Previous Article | Next Article 
The Journal of Neuroscience, March 1, 2003, 23(5):1730
Glycoprotein 130 Signaling Regulates Notch1
Expression and Activation in the Self-Renewal of Mammalian Forebrain
Neural Stem Cells
Andrew
Chojnacki1,
Takuya
Shimazaki1,
Christopher
Gregg1,
Gerry
Weinmaster2, and
Samuel
Weiss1
1 Genes & Development Research Group, Department of
Cell Biology and Anatomy, University of Calgary, Faculty of Medicine,
Calgary, Alberta, Canada T2N 4N1, and 2 Department of
Biological Chemistry, Molecular Biology Institute, University of
California at Los Angeles School of Medicine, Los Angeles, California
90095-1737
 |
ABSTRACT |
Glycoprotein130 (gp130) and Notch signaling are thought to
participate in neural stem cell (NSC) self-renewal. We asked whether gp130 regulates Notch activity in forebrain epidermal growth factor (EGF)-responsive NSCs. Disruption of Notch1 using
antisense or a 
-secretase inhibitor demonstrated a requirement for
Notch1 in the maintenance and proliferation of NSCs.
Ciliary neurotrophic factor (CNTF) activation of gp130 in NSCs rapidly
increased Notch1 expression. NOTCH1 activation,
indicated by tumor necrosis factor 
-converting enzyme (TACE)-
and presenilin-mediated processing, also
increased. Infusion of EGF+CNTF into adult forebrain lateral ventricles
increased periventricular NOTCH1 compared with EGF alone. Neither
Hes1 (hairy and enhancer of
split) nor Hes5 appeared to mediate
gp130-enhanced NOTCH1 signaling that regulates NSC maintenance. This is
the first example of a link between gp130 signaling and NOTCH1 in
regulating NSC self-renewal.
Key words:
notch; gp130; CNTF; stem cell; delta; self-renewal
| |
Introduction |
Two principal
characteristics of neural stem cells (NSCs) are multipotency and
self-renewal, the ability to maintain this multipotency after repeated
rounds of proliferation (for review, see Gage, 2000
; Alvarez-Buylla et
al., 2000). In the adult mammalian CNS, a population of NSCs reside in
the periventricular area of the forebrain lateral ventricles (Reynolds
and Weiss, 1992; Morshead et al., 1994) and contribute neurons to the
olfactory bulb throughout adulthood (Lois and Alvarez-Buylla, 1994;
Doetsch and Alvarez-Buylla, 1996). It is likely that epidermal growth
factor (EGF) or transforming growth factor (TGF) is the in
vivo mitogen for adult forebrain NSCs (Reynolds et al., 1992;
Morshead et al., 1994; Tropepe et al., 1997; Doetsch et al., 1999). On
the other hand, little is known about the epigenetic regulation of NSC
self-renewal.
Glycoprotein130 (gp130) mediates signaling initiated by the cytokine
class of secreted factors, which include leukemia inhibitory factor
(LIF), ciliary neurotrophic factor (CNTF), oncostatin M, and
interleukin-6 (IL-6), among others (for review, see Turnley and
Bartlett, 2000). LIF signals through the dimerization of its cognitive
receptor, LIF receptor 
(LIFR), with gp130, whereas CNTF
signaling is mediated by a heterotrimeric complex consisting of CNTF
receptor (CNTFR), LIFR, and gp130 subunits. The long-term maintenance of embryonic stem (ES) cells and NSCs requires the presence
of LIF or CNTF (Smith et al., 1988; Williams et al., 1988; Conover et
al., 1993; Carpenter et al., 1999; Shimazaki et al., 2001). We recently
reported that CNTF signaling (through the CNTF/LIF/gp130 receptor
complex) acts to maintain embryonic and adult NSCs in an
undifferentiated state by blocking NSC differentiation to restricted
glial precursors, with no action on stem cell survival or proliferation
(Shimazaki et al., 2001). The mechanisms underlying the actions of CNTF
on NSC self-renewal are not understood.
Notch signaling has also been implicated in NSC maintenance (for
review, see Artavanis-Tsakonas et al., 1999). Deletion of the basic
helix-loop-helix (bHLH) transcriptional repressor Hes1 (hairy and enhancer of split), a known
mediator of Notch signaling, causes premature neuronal progenitor cell
differentiation and a reduction in the self-renewal capacity of
embryonic forebrain NSCs (Nakamura et al., 2000). Overexpression of
activated NOTCH1 in the embryonic cortex results in an increase of
radial glial cells (Gaiano et al., 2000), which have been implicated in
neurogenesis (for review, see Alvarez-Buylla et al., 2001). Recently,
Hitoshi et al. (2002) demonstrated that Notch signaling was required
for the maintenance of NSCs but not their generation; however, Notch signaling appears to be context dependent and can also promote glial
cell fate (Morrison et al., 2000; Chambers et al., 2001).
Given the context-dependent nature of Notch signaling, we first
tested whether it functioned in the maintenance of NSCs derived from
the basal forebrain. We then tested the hypothesis that gp130 signaling
regulates NSC self-renewal by regulating Notch signaling. Our in
vitro and in vivo data support the conclusions that
NOTCH1 signaling functions in NSC maintenance and proliferation and
that activation of gp130 leads to an increase in NOTCH1 signaling.
| |
Materials and Methods |
Animals and genotyping. Breeding and genotyping of
LIFR mice has been described previously (Shimazaki et al., 2001). CD1
mice were obtained from the University of Calgary Animal Resources Center (Calgary, Alberta, Canada).
Cell culture. Generation of primary and secondary embryonic
day 14 (E14) striatal neurospheres was performed as described previously (Shimazaki et al., 2001). Briefly, dissociated primary neurospheres were cultured at a density of 0.05 × 106 cells/ml in culture flasks containing
either EGF alone or EGF and rat CNTF [20 ng/ml; Peprotech
(Rocky Hill, NJ) and gift from Dr. Rob Dunn (McGill University),
respectively], unless stated otherwise. Additionally, IL-6 and soluble
IL-6 receptor (sIL6R) (both from R&D, Minneapolis, MN) were used
at 20 and 25 ng/ml, respectively. Cells were cultured for a maximum of
7 d in vitro (DIV) and harvested for various molecular
and biochemical analyses stated below. For NOTCH1 immunoreactive cell
counts, primary neurospheres were dissociated and cultured at 50,000 cells/ml for 6 hr in either EGF or EGF+CNTF on
poly-L-ornithine-coated coverslips and processed for NOTCH1 immunocytochemistry as stated below.
RT-PCR-Southern blot. Total RNA was isolated from
neurospheres using Trizol reagent (Invitrogen, Carlsbad,
CA). First-strand cDNA was synthesized using Superscript RT
(Invitrogen) at an incubation time of 75-90 min at
42°C. RT-PCR analysis was used to establish the presence of
Notch1, -2, -3, -4,
Delta1 and -3, Jagged1 and -2, Hes1 and -5, and Mash1
in EGF-derived stem cell progenies using the conditions stated in Table
1. Each product was amplified by
denaturation (94°C, 45 sec), primer annealing (45 sec), and extension
(72°C 45 sec; Jagged 2, Notch1, and
Notch4 for 1 min) with the exception of Delta1,
which was a two-step PCR (94°C for 45 sec denaturation and anneal at
72°C for 1 min). Identity of amplified products was established by
Southern blot analysis using Notch1, Delta3,
Jagged1, Jagged2, and Delta1 (kindly
provided by Dr. Domingos Henrique, Lisbon Medical School) cDNA probes
or by PCR-based direct cloning and sequencing of Notch2,
-3, -4, Jagged1, Hes1,
Hes5, and Mash1. PCR products were purified using the Geneclean II kit (BIO 101) and ligated into pGEM-T
vector plasmids (Promega, Madison, WI). Sequencing
identified correct plasmid clones. Southern blot analysis was performed
as described previously (Shimazaki et al., 1999). Experiments were
performed at least three times with the exception of RT-PCR analyses,
which were performed twice. Pictures were taken on a Kodak
DC120 (Rochester, NY) and densitometric analysis was done using
Kodak Digital Science 1D software.
Western blotting. Cultured cells were processed for Western
blot analysis as described previously (Shimazaki et al., 1999). NucliePURE prep kit (Sigma, St. Louis, MO) was used for
the isolation of nuclear proteins as per the manufacturer's
instructions. Nitrocellulose membranes were incubated with the 93-4 rabbit mouse NOTCH1 primary antibody (1:10,000), or
affinity-purified (AFP) goat mouse NOTCH1 antibody (1:100;
Santa Cruz Biotechnology, Santa Cruz, CA), or mouse mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute
of Technology), and/or AFP goat ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and
then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon,
Temecula, CA). Blots were developed using Enhanced Chemiluminescence
and Hyperfilm (both from Amersham Biosciences, Baie
d'Urfé, Quebec). Pictures and analysis were done as above.
Immunohistochemistry. Mice were processed for
immunohistochemistry as described previously (Shimazaki et al., 2001).
Coronal cryosections (8 µm) of mouse forebrain were double stained
for Notch1 and CNTFR as described below. Embryonic sections were
postfixed with 100% acetone for 30 sec, preblocked with rabbit IgG
Fab2 fragment (Jackson ImmunoResearch, West Grove, PA;
1:100 in 10% normal donkey serum, 0.4% Triton X-100, PBS, pH
7.5) for 2 hr at room temperature, incubated overnight at 4°C with
goat anti-rat CNTFR IgG (1:100; Santa Cruz Biotechnology), washed
with PBS, incubated for 1 hr at room temperature with biotin-conjugated donkey goat IgG secondary (1:200; Jackson
ImmunoResearch), followed by a 1 hr incubation in
streptavidin-Cy3 (1:1000; Jackson ImmunoResearch). Sections were then washed with PBS and incubated overnight at 4°C
with rabbit 93-4 anti-rat NOTCH1 (1:25). Sections were then washed and
incubated with Hoechst 33258 and goat anti-rabbit fragment crystallisable-specific (1:100; FITC conjugated) secondary
antibody for 30 min at 37°C, washed, and mounted with FluorSave
(Calbiochem, San Diego, CA). Adult sections were incubated
overnight at 4°C with rabbit anti-mouse NOTCH1EC (1:50; EC
indicates extracellular antibody directed against the extracellular
portion of NOTCH1 as named by Dr. Lendahl; gift from Dr. Urban Lendahl,
Karolinska Institute) in 5% NGS, washed with PBS, incubated with
biotin-conjugated goat anti-rabbit IgG for 1 hr at room temperature
(1:200; Jackson ImmunoResearch), followed by a wash with
PBS, incubated with streptavidin-Cy3 as above. Images were taken on a
Photometrics Quantix camera (Tucson, AZ) mounted on a
Zeiss Axioplan2 (Thornwood, NY) or with a
Cohu CCD (San Diego, CA) mounted on a Zeiss
Axiovert (for time-lapse images).
Counts of NOTCH1-immunoreactive cells in vitro and
in vivo. Dissociated primary spheres, were exposed to either EGF
or EGF+CNTF for 6 hr on poly-L-ornithine-coated coverslips.
The cells were fixed for 20 min with 4% paraformaldehyde at room
temperature. Cells were then preblocked in 10% NGS for 1 hr, incubated
with NOTCH1EC at 1:2000 overnight at 4°C, and then incubated with
rhodamine-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch). Pictures of five random fields of each
condition per independent experiment were taken on a
Photometrics Quantix camera mounted on a
Zeiss Axioplan2. To capture images of NOTCH1
immunoreactivity, all fields were exposed for 1.5 sec. Pictures were
imported into Adobe Photoshop 4.0. To aid in the distinction between
weakly and intensely staining NOTCH1 cells, the brightness was reduced
by 150% in all cases. Blind counts were made on the percentage of
intensely immunoreactive NOTCH1-positive cells out of total
Hoechst 33258 positive cells. For counts of the number of
NOTCH1-immunoreactive cells in EGF- and EGF+CNTF-infused animals, six
sections (from three independent experiments of EGF and EGF+CNTF
infusions) were randomly selected at approximately the same
rostral-caudal level, and images of NOTCH1 and Hoechst immunolabeling
were captured as above, imported, and pseudocolored in Photoshop 4.0. Respective NOTCH1 and Hoechst panels were overlapped, and 21.6 µm
(medial-lateral) × 173 µm (dorsal-ventral) of the expanded
lateral ventricle immediately below the dorsal limit was outlined and
counted double blind for the number of NOTCH1-immunoreactive cells.
Notch1 antisense and -secretase
inhibitor. Oligonucleotides were designed against a portion of the
5' intracellular cdc10/ankyrin repeat region (CDC) as described
previously (Austin et al., 1995). The CDC antisense sequence
5'-CCTCCACTGCAGGAGGCAATCAT-3' was identical to the
one described previously with the exception of a G-A (in bold)
switch in the mouse sequence. Antisense oligonucleotides were used in
parallel with their corresponding sense oligonucleotides at 20 µM. Briefly, oligonucleotides were added to 2 million dissociated pass 1 (P1) cells in 5 ml of EGF media. Cells were
triturated with a fire-polished Pasteur pipette and moved into flasks
(Falcon, BDL, Franklin Lakes, NJ); 6 hr later flasks were tapped until cells lifted off the plastic surface. Twenty-four hours later, 4 ml of
cells was harvested for Western blot analysis, and the remaining 1 ml
was transferred to a six-well plate, and allowed to grow for 3 DIV;
individual spheres were dissociated in 96-well plates and assayed for
the ability to produce secondary spheres after 7 DIV. For the
-secretase inhibitor (Calbiochem) experiments, 50 µM of the inhibitor was added to 2 million
dissociated P1 cells in 5 ml of EGF media, allowed to grow for 24 hr,
and then harvested for Western blot analysis. For the detection of
NOTCH1-protein fragment 2 (PF2), 2 million dissociated P1 cells
in 5 ml of EGF were allowed to grow for 24 hr, treated with DMSO or
-secretase inhibitor (50 µM) for 4 hr, and
then harvested for Western blot analysis. For the detection of
NOTCH1-PF3, P1 cells were cultured for 3 DIV at a concentration of
400,000 cells/ml, and then DMSO or -secretase inhibitor II (50 µM) was added for 4 hr; the cells were then
isolated for nuclear proteins as stated above. For the single-sphere
dissociation experiments, -secretase II was added to a concentration
of 30 µM, at plating, to dissociated P1 cells in a 24-well plate at a concentration of 400,000 cells/ml. Neurospheres were grown for 3 DIV, and then individual spheres were dissociated in
96-well plates and assayed for secondary neurosphere production. DMSO
added to control cultures was equal to the volume of -secretase II
inhibitor added.
In vivo growth factor infusions. In vivo
infusion of EGF and EGF+CNTF were performed as described in Shimazaki
et al. (2001).
| |
Results |
Notch1 signaling is required for the maintenance of E14
EGF-responsive NSCs
EGF-responsive NSCs of the basal forebrain proliferate to form
neurospheres, which contain precursors to neurons, astrocytes, and
oligodendrocytes (Reynolds and Weiss, 1996). The in vitro maintenance of an undifferentiated state by NSCs may be studied through
the ability of single EGF-generated neurospheres, which are dissociated
and cultured in the presence of EGF, to give rise to secondary
neurospheres (Reynolds and Weiss, 1996). We recently found that the
CNTFR/LIFR/gp130 receptor complex operates in the maintenance of
EGF-derived NSCs (Shimazaki et al., 2001). Specifically, when single P1
neurospheres (P1 neurospheres are derived from dissociated primary
neurospheres, which in turn are derived from the culture of dissociated
E14 striatopallidum complexes in the presence of EGF) generated in the
presence of EGF+CNTF were individually dissociated and replated in EGF
alone, they produced 59% more pass 2 (P2) neurospheres than
equivalent-sized P1 neurospheres generated in EGF and replated in EGF.
Before asking whether CNTF could modulate NOTCH1 signaling, we asked
whether Notch1 mediates, at least in part, the maintenance
of EGF-responsive NSCs. We analyzed the ability of EGF-generated P1
neurospheres to produce P2 neurospheres after culturing them in the
presence of a well characterized antisense to the CDC repeat
portion of Notch1 (Austin et al., 1995;
Redmond et al., 2000). Exposure of dissociated primary
neurospheres to Notch1 antisense (20 µM) for their first 24 hr in culture resulted
in a significant decrease in NOTCH1 expression, when compared with
cells exposed to sense controls (Fig.
1A). Furthermore,
antisense-treated P1 neurospheres had a significantly reduced capacity
to produce P2 neurospheres compared with sense controls, whether
assayed by single-sphere dissociation of equivalent-sized neurospheres
(43%) (Fig. 1A) or batch culture experiments (40%)
(Fig. 1B). These results suggest that NOTCH1
expression levels can regulate the maintenance of EGF-responsive
NSCs.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1.
Notch1 antisense reduces NOTCH1 expression and NSC
self-renewal. NSCs were cultured in the presence of 20 ng/ml EGF in the
absence or presence of 20 µM Notch1
antisense and harvested after 1 DIV for protein or cultured for a total
of 3 DIV (to form P1 neurospheres) and assayed either by single-sphere
dissociation (A) or batch culture
(B) for the formation of P2 neurospheres.
A, Western blot analysis reveals a reduction in
NOTCH1-PF1 expression (inset; p < 0.05; t test; n = 3) in
antisense-treated P1 neurospheres. A concomitant decrease was observed
in the ability of antisense-treated, individual equivalent sized P1
neurospheres to produce P2 neurospheres (*p < 0.05; t test; n = 3) compared with
sense treatment. B, Assaying for the ability of P1
neurospheres treated with Notch1 antisense to produce P2
neurospheres by batch culture analysis also reveals a significant
decrease in their ability to produce P2 neurospheres
(*p < 0.05; t test;
n = 3).
|
|
We next sought to determine whether NOTCH1 cleavage/activation is
necessary for the maintenance of EGF-responsive NSCs. A -secretase-like protease has recently been implicated in the cleavage of NOTCH1 into its active intracellular domain (De Strooper et
al., 1999) and can be blocked by the peptidomimetic inhibitor -secretase inhibitor II (De Strooper et al., 1999; Wolfe et al., 1999). We use the nomenclature for NOTCH1 processing and products defined by Mumm et al. (2000) and Brou et al. (2000). Therefore, processing of NOTCH1 by a furin-like convertase at the S1 site produces NOTCH1-PF1 (Logeat et al., 1998), ligand-dependent processing at the S2 site by TACE produces NOTCH1-PF2, and ligand-dependent processing at the S3 site by a presenilin-mediated cleavage
produces the active intracellular portion of NOTCH1 or NOTCH1-PF3 (De
Strooper et al., 1999). Inhibition of -secretase should result in
the accumulation of NOTCH1-PF2 if the inhibitor is effective (Brou et
al., 2000; Mumm et al., 2000). Therefore, we tested the effectiveness of -secretase inhibitor II in preventing the production of
NOTCH1-PF3 by assaying for the accumulation of NOTCH1-PF2 (which is
expected because NOTCH-PF2 is the precursor for NOTCH1-PF3) and for the decrease in NOTCH1-PF3 in EGF-generated neurospheres. Western blot
analysis of P1 neurospheres treated for 4 hr with -secretase inhibitor II (50 µM) consistently revealed the
appearance of a band (NOTCH1-PF2; n = 4) below that of
NOTCH1-PF1, compared with EGF and EGF+DMSO controls, suggesting that
the inhibitor was preventing the production of NOTCH1-PF3 (Fig.
2A). Furthermore,
nuclear protein extracts of P1 neurospheres that were treated for 4 hr
with -secretase inhibitor II revealed a decrease (relative to
HISTONE H1 expression: 
34% in experiment 1, 27% in
experiment 2) in NOTCH1-PF3 compared with DMSO controls, confirming
that the inhibitor was preventing the production of NOTCH1-PF3 (Fig.
2B). Addition of -secretase inhibitor (30-50
µM), at plating, to a single-cell suspension derived from primary neurospheres, delayed the formation of P1 neurospheres by ~24 hr, compared with vehicle controls (Fig.
2C-H). Once generated, inhibitor-treated P1
neurospheres appeared more differentiated, compared with vehicle
controls (Fig. 2, compare G, H). Western
blot analysis revealed that 24 hr after inhibitor addition, NOTCH1-PF1
protein expression was reduced to 52% of vehicle-treated sister
cultures (n = 3, p < 0.005) (Fig.
2I), suggesting that inhibition of NOTCH1 activation
leads to an overall decrease in NOTCH1 production. After 3 DIV,
-secretase inhibitor- and vehicle-treated P1 neurospheres of
150-200 µm in diameter were isolated, dissociated, and examined for
the formation of P2 neurospheres. -secretase inhibitor treatment
reduced P2 neurosphere formation to 61% of control (n = 3, p < 0.05) (Fig. 2I). These results suggest that, in addition to expression levels, NOTCH1 cleavage
and signaling regulate the maintenance of EGF-responsive NSCs.
View larger version (42K):
[in this window]
[in a new window]
|
Figure 2.
Disruption of NOTCH1 signaling by -secretase
inhibitor II delays P1 neurosphere formation and reduces their ability
to produce P2 neurospheres. A, To ensure that the
-secretase inhibitor that we were using was effectively blocking
production of NOTCH1-PF3, NSCs were cultured in 20 ng/ml EGF (20 ng/ml)
for 24 hr, at which point DMSO (carrier) or -secretase inhibitor II
(50 µM) was added, and the cells were harvested 4 hr
later for total proteins and Western blot analysis. A,
The asterisk indicates an increase in the P2 proteolytic
product of NOTCH1, as would be expected if the -secretase inhibitor
was effectively blocking production of NOTCH1-PF3
(n = 3), and identifies the upper band as
furin-processed NOTCH1 or NOTCH1-PF1. B, Three day
in vitro P1 neurospheres that were treated with
-secretase inhibitor for 4 hr and harvested for nuclear proteins and
Western blot analysis demonstrate a decrease in NOTCH1-PF3 compared
with DMSO control. C-H, NSCs were cultured in EGF (20 ng/ml) and either DMSO (C, E,
G; carrier) or -secretase inhibitor II
(D, F, H; 30 µM), and digital micrographs were taken after 6 (C, D), 18 (E,
F), and 88 hr (G,
H). I, Single-sphere dissociation
assay reveals a significant reduction in self-renewal capacity of P1
neurospheres generated for 3 DIV in the presence of -secretase inhibitor II (30 µM) compared with DMSO controls (*p < 0.05; t test; n = 3).
Inset shows a reduction of NOTCH1-PF1 expression in P1
neurospheres treated for 1 DIV, from the time of plating, with 50 µM -secretase inhibitor II compared with the DMSO
control (p < 0.05; t test;
n = 3), indicating that constitutive inhibition of
NOTCH1 activation for at least 24 hr leads to an overall decrease in
NOTCH1 expression. Scale bar, 100 µm. N.S.,
Nonspecific; -SI, -secretase inhibitor.
|
|
Signaling through CNTFR regulates expression of Notch1
in vitro
Given the similarity between the actions of NOTCH1 and
gp130-mediated signaling on NSC maintenance, we asked whether
gp130-mediated signaling, stimulated by CNTF, could regulate NOTCH1
expression in EGF-responsive NSCs. We first explored whether CNTFR
and NOTCH1 are coexpressed in the developing E14 basal forebrain, the
origin of embryonic EGF-responsive NSCs. We found that most of the
NOTCH1-expressing cells coexpress CNTFR in the E14 basal forebrain
germinal zone (Fig. 3A-D),
consistent with the hypothesis of a link between CNTFR and NOTCH1
signaling in forebrain NSCs. We then examined the results of gp130
activation on NOTCH1 signaling in EGF-generated neurospheres. When
screening, using RT-PCR, for Notch gene expression, we found
that P1 neurospheres expressed Notch1 and Notch3
but not Notch2 or Notch4 (data not shown).
Quantitative RT-PCR Southern blot analysis was used to examine
Notch expression in P1 neurospheres generated in EGF+CNTF
(20 ng/ml) compared with those generated in EGF. Notch1
increased approximately threefold in 1 DIV EGF+CNTF-generated P1
neurospheres compared with EGF-generated P1 neurospheres (Fig. 3E), whereas Notch3 expression was unaffected
(Fig. 3F). Because increases in mRNA expression are
not always followed by concomitant increases in protein expression, we
sought definitive evidence that gp130-mediated signaling could regulate
NOTCH1 expression. We used two antibodies to NOTCH1 to ensure that we
were in fact measuring bona fide NOTCH1 protein. Figure
3G shows that in a Western blot, both the 93-4 (Shawber et
al., 1996) and Santa Cruz M-20 antibodies identify increases in
NOTCH1-PF1 and NOTCH1-PF2 (more than fivefold; p < 0.01; n = 5) in 3 DIV EGF+CNTF-generated P1
neurospheres compared with EGF-generated P1 neurospheres. Furthermore, nuclear protein extracts of 3 DIV EGF+CNTF-treated P1 neurospheres demonstrate an increase in NOTCH1-PF3 compared with EGF controls as
determined by Western blot analysis (Fig. 3H)
(p < 0.01; t test; n = 4). Together, these findings suggest that the increase in NOTCH1
synthesis in CNTF-stimulated P1 neurospheres further results in
ligand-mediated activation of NOTCH1 signaling.
View larger version (61K):
[in this window]
[in a new window]
|
Figure 3.
EGF+CNTF treatment of embryonic P1 neurospheres
specifically increases Notch1 mRNA and protein
expression. A-D, Immunofluorescence micrographs of a
coronal section (8 µm) through the forebrain of an E14 mouse embryo.
A, Nuclei were labeled with Hoechst 33258 (blue). CNTFR-immunoreactive cells in the ventricular
zone were visualized with Cy3 (B, red),
and Notch1-immunoreactive cells were labeled with FITC
(C, green). D, A merged
image of B and C, where yellow staining
indicates colocalization of NOTCH1 and CNTFR. Box in
A indicates area magnified in B-D.
E-G, NSCs were cultured in 20 ng/ml EGF, the absence or
presence of 20 ng/ml CNTF, and harvested after 24 hr for total RNA and
RT-PCR Southern blot analysis (E,
F), or after 3 DIV for Western blot analysis
(G). Notch1 expression increased
significantly (*p < 0.05 vs EGF; t
test; n = 3) after 1 DIV of CNTF treatment
(E) compared with no change in
Notch3 expression (F). Both the
93-4 and Santa Cruz intracellular NOTCH1 antibodies reveal an increase
in NOTCH1-PF1 and NOTCH1-PF2 proteolytic products after 3 DIV of
EGF+CNTF treatment compared with EGF alone (G)
(p < 0.01; t
test; n = 5). Nuclear expression of NOTCH1-PF3
increases in 3 DIV P1 neurospheres cultured constitutively in EGF+CNTF
compared with EGF alone (H)
(p < 0.01; t test;
n = 4). Scale bars: A, 50 µm;
D, 100 µm. LGE, Lateral ganglionic
eminence; LV, lateral ventricle; CTX,
cortex.
|
|
Lateral inhibition is not necessary for CNTF to increase NOTCH1
expression; however, cell-cell contact is required for CNTF to
increase NSC self-renewal
To determine whether CNTF increases Notch1 expression
directly or indirectly, we examined the time course of CNTF-induced NOTCH1 expression. NOTCH1 expression increases significantly in 3 DIV
neurospheres after as little as 2 hr of exposure to CNTF (Fig.
4A). These data suggest
that de novo synthesis of another protein, which would then
act to increase expression of NOTCH1, is unlikely. In all of the above
mentioned experiments, we examined Notch1 mRNA and protein
expression in developing clusters of cells (neurospheres). Given the
cell-cell contact within neurospheres, it is possible that lateral
inhibition mediates the actions of CNTF on Notch1
expression. In this case, CNTF could decrease ligand expression, which
through lateral inhibition would increase Notch1 expression
in the same population of cells. To examine this possibility, we tested
whether 6 hr of EGF+CNTF exposure (20 ng/ml), in comparison with EGF
alone, could increase NOTCH1-PF1 or NOTCH1-PF2 expression in a
completely dissociated single-cell suspension (5 × 104 cells/ml) derived from 7 DIV primary
neurospheres. Western blot analysis shows that in the absence of
cell-cell contact, CNTF can increase NOTCH1-PF1 expression (more than
threefold; p < 0.05; t test;
n = 3) (Fig. 4B). We did not detect
NOTCH1-PF2 in either condition, which is what we would have predicted
considering that this band should appear only in the presence of
ligand-mediated activation of TACE and further indicates that we have
correctly identified NOTCH1-PF2. These data suggest that the
CNTF-induced increase in NOTCH1 expression is ligand independent but
that NOTCH1 activation requires ligand mediated cleavage.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 4.
Cell-cell contact is not required for CNTF to
increase NOTCH1 expression but is required for CNTF to increase NSC
self-renewal. A, Western blot analysis reveals that
NOTCH1-PF1 expression increases as early as 2 hr after CNTF treatment
of 3 DIV EGF-derived P1 neurospheres (n = 3).
B, Totally dissociated primary neurospheres were
cultured in EGF+CNTF for 6 hr; Western blot analysis demonstrates a
threefold increase in NOTCH1-PF1 expression
(p < 0.05; t test;
n = 3) compared with EGF controls. No increase in
NOTCH1-PF2 could be detected. C, P1 neurospheres were
generated in EGF or in EGF+CNTF in the absence (0-24 hr) or presence
(72-96 hr) of cell-cell contact. After 7 DIV the three different
groups were assayed for the formation of P2 neurospheres by
single-sphere dissociation and culture in EGF alone (each group was
washed at 24 and 96 hr). Compared with EGF, addition of CNTF for 24 hr
at 3 DIV increased the formation of P2 neurospheres by 59%
(p < 0.0001; Tukey HSD test;
n = 3), whereas there was no difference in P2
neurosphere formation when CNTF was added for the first 24 hr
(p > 0.58; Tukey HSD test;
n = 3).
|
|
We then tested whether an increase in NOTCH1 expression, without its
activation, was sufficient to increase the production of P2
neurospheres. Thus we treated P1 cells with EGF alone or with EGF and
then added CNTF for 24 hr at plating or for 24 hr at 3 DIV. All
conditions were washed at 1 and 4 DIV. After 7 DIV, we dissociated
single P1 neurospheres to assay for self-renewal (by counting the
numbers of P2 neurospheres per single P1 neurosphere). Figure
4C shows that there was no significant increase in
self-renewal of P1 neurospheres that were treated with CNTF for the
first 24 hr, whereas there was a significant increase in self-renewal
capacity in the P1 neurospheres treated with CNTF at 3 DIV
[p < 0.0001; Tukey honestly significant
difference (HSD) test; n = 3]. These data
suggest that cell-cell contact, which is present in 3 DIV spheres and
almost entirely absent in plated cells (for the first 24 hr at this
concentration), is necessary for CNTF to increase the self-renewal
capacity of NSCs.
Although we had shown that CNTF could increase NOTCH1 expression in
EGF-generated neurospheres, we had yet to demonstrate directly the
phenomenon at the single-cell level. With this in mind and to determine
whether CNTF increases expression of NOTCH1 in all EGF-generated cells
or rather increases the number of NOTCH1-expressing cells, we examined
the expression of NOTCH1 in dissociated primary spheres (P1 cells)
treated for 6 hr with either EGF or EGF+CNTF. We observed that all of
the cells, in either condition, expressed some level of
immunoreactivity to NOTCH1 (data not shown). We found, however, that
addition of CNTF to the culture increased the number of cells that
labeled intensely (Fig. 5,
arrowheads) (see Materials and Methods for experimental
details) compared with those that labeled weakly (Fig. 5, small
arrows) for NOTCH1 by 49% (p < 0.003;
t test; n = 3) compared with EGF alone.
Taken together, these findings suggest that CNTF directly upregulates NOTCH1 expression in EGF-generated NSC progeny.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 5.
CNTF increases the number of intensely
NOTCH1-immunoreactive cells. Primary neurospheres were dissociated and
then cultured on poly-L-ornithine-coated coverslips for 6 hr in either EGF (A, B) or EGF+CNTF
(C, D), and blind counts were made (as
described in Materials and Methods) on the number of intensely
NOTCH1-immunoreactive cells (C).
A, C, Nuclei were labeled using Hoechst
33258 (blue). NOTCH1-immunoreactive cells were labeled
with rhodamine (B, D,
red). E, Compared with cells cultured in
EGF alone, cells cultured in EGF+CNTF demonstrate a 49% increase in
the number of intensely NOTCH1-immunoreactive cells
(*p < 0.003; t test;
n = 3). Arrowheads indicate examples
of cells that stain intensely for NOTCH1, and small
arrows indicate examples of cells that stain weakly for NOTCH1.
Scale bar, 20 µm.
|
|
NOTCH1 expression correlates with the capacity of P1 neurospheres
to generate P2 neurospheres in LIFR knock-out mice
We have recently reported that adult LIFR
heterozygotes show a decrease in the ability to produce forebrain
neurospheres (indicative of NSC number), compared with their wild-type
littermates (Shimazaki et al., 2001). To determine whether gp130
regulation of NOTCH1 function is associated with changes in NSC
self-renewal, we compared NOTCH1 protein expression, using Western blot
analysis, in 3 DIV LIFR+/+
and
LIFR
/
embryonic P1 neurospheres treated with EGF or EGF+CNTF. Furthermore, we
examined the capacity of wild-type and mutant P1 neurospheres to
produce P2 neurospheres by single sphere dissociation. In wild-type (+/+) P1 neurospheres, both NOTCH1-PF1 and -P2 neurosphere production increase after CNTF addition, whereas no increase in NOTCH1-PF1 or -P2
neurosphere production was observed in
LIFR/
P1 neurospheres cultured in EGF+CNTF (Fig.
6). Glycoprotein130 signaling through
IL6R does not require LIFR and thus provides a means to test whether
gp130 activation is sufficient for increasing NOTCH1-PF1 expression and
P2 neurosphere production. Because the IL6 receptor is not
expressed in EGF-generated neurospheres (T. Shimazaki and S. Weiss,
unpublished observations), we generated LIFR/
P1 neurospheres in the presence of sIL6R+IL6. Activation of gp130 signaling in
LIFR/
P1 neurospheres with sIL6R+IL6 was sufficient to increase their expression of NOTCH1-PF1 and -P2 neurosphere production (Fig. 6).
These experiments suggest that activation of gp130 signaling is the
common element in CNTFR-, LIFR- and IL6R-mediated increases in
NOTCH1 expression of P1 neurospheres and P2 neurosphere production.
View larger version (33K):
[in this window]
[in a new window]
|
Figure 6.
IL6+sIL6R increases NOTCH1 expression and P2
neurosphere production in P1 neurospheres generated from
LIFR/
mice. P1 neurospheres were generated from wild-type (+/+) or null
mutant (/) LIFR littermates, in the various
conditions indicated, and were then assayed after 3 DIV for NOTCH1
protein with Western blot and after 7 DIV for P2 neurosphere production
by single-sphere dissociation in EGF alone. Increase in P2 neurosphere
production in wild-type (+/+) EGF+CNTF generated P1 neurospheres and in
LIFR/
P1 neurospheres generated in EGF+IL6+sIl6R correlated with concomitant
increases in NOTCH1-PF1 expression (inset). CNTF had no
effect on P2 neurosphere production or NOTCH1-PF1 expression in
LIFR/
P1 neurospheres. **p < 0.01 versus +/+ control
culture or / control culture; Tukey HSD test; n = 5. N-PF1, NOTCH1-PF1.
|
|
The CNTF-induced increase in NOTCH1 expression is
context dependent
In vitro, one can determine the effects of
factors on a population of cells one at a time or in combination. This
is certainly not the case in the developing germinal zone, where
progenitor cells are likely exposed to several factors at the same
time. Thus, we sought to determine whether CNTF could increase
NOTCH1-PF1 expression in the absence of EGF. We stimulated dissociated
7 DIV primary neurospheres (5 × 104
cells/ml) with CNTF for 6 hr, in the absence or presence of EGF (Fig.
7A). In the absence of EGF,
CNTF failed to increase NOTCH1-PF1 expression. These data suggested
that only cells receiving an EGF signal could respond to CNTF with an
increase in NOTCH1 expression. An alternative possibility is that a
proliferative state is necessary for CNTF to increase NOTCH1 expression
in NSCs. Thus, we tested whether CNTF could increase NOTCH1-PF1
expression in the absence of EGF but in the presence of FGF2, another
principal NSC mitogen. Figure 7B demonstrates a significant
increase in NOTCH1-PF1 expression in a single-cell suspension derived
from 7 DIV primary neurospheres cultured for 24 hr in FGF2+CNTF,
compared with those exposed to FGF2 alone. Therefore, induction of
NOTCH1 expression by CNTF appears to require that NSCs be in a
proliferative state.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 7.
The CNTF-induced increase in NOTCH1
expression in dissociated primary neurospheres is dependent on either
EGF or FGF2 signaling. A, B, Single-cell
suspensions derived from primary neurospheres and cultured for 6 hr in
the indicated conditions reveal that CNTF had no effect on NOTCH1-PF1
expression in the absence of EGF (B)
(n = 3) and that CNTF can increase
NOTCH1-PF1 expression in either EGF- or FGF2-containing media
(B) (p < 0.05; t test; n = 3).
|
|
CNTF can upregulate NOTCH1 expression
in vivo
Intraventricular infusion of EGF+CNTF resulted in a 50% increase
in the number of neurospheres that could be derived from the
periventricular area of the adult brain, compared with EGF infusion
alone (Shimazaki et al., 2001). Given that CNTF could only induce the
expression of NOTCH1 in the presence of either EGF or FGF2 (Fig. 7), we
compared NOTCH1 expression in the forebrain periventricular area of
EGF- versus EGF+CNTF-infused adult mice. Adult CD1 mice were infused
with EGF or EGF+CNTF for 6 d, and the brains were processed for
NOTCH1 immunohistochemistry (n = 3 for each treatment).
The lateral aspect of the periventricular area (the specific region
that is thought to be enriched in NSCs) of brains infused with EGF+CNTF
exhibited a much thicker and more intense area of NOTCH1 expression
compared with animals infused with EGF alone (Fig.
8, compare A, B).
In particular, although NOTCH1 immunoreactivity is sporadic on the
lateral aspect of the ventricle in the EGF infused brain, NOTCH1
staining appears as a thick, continuous layer in the
EGF+CNTF-infused brain (Fig. 8, compare C, E). We
then performed double-blind counts on the number of cells
immunoreactive for NOTCH1 in the EGF- and EGF+CNTF-infused animals. Of
the cells within theexpanded lateral ventricular area, 76 ± 12%
expressed NOTCH1 in animals infused with EGF+CNTF compared with 39 ± 7% in the EGF-infused animals (p < 0.026;
n = 3 each group; t test). Thus,
CNTF, in the presence of EGF, can upregulate the number of
NOTCH1-immunoreactive cells in vivo.
View larger version (145K):
[in this window]
[in a new window]
|
Figure 8.
CNTF enhances the expression of NOTCH1 in
vivo. Adult CD1 mice were infused with either EGF (A,
C, D) or EGF+CNTF (B, E,
F) for 6 d, after which brains were processed for
NOTCH1 immunohistochemistry. Infusion of EGF+CNTF resulted in an
overall increase in NOTCH1 staining intensity as well as a markedly
thickened layer of NOTCH1 expression on the lateral aspect
(A, B, arrows) of the
ventricle. Furthermore, more cells in the ventricular zone labeled for
NOTCH1 in EGF+CNTF (C, NOTCH1, 76 ± 12%;
D, Hoechst) compared with EGF (E, NOTCH1,
39 ± 7%; F, Hoechst) infused mice
(p < 0.026; t test;
n = 3 each group). C-F,
Arrows indicate NOTCH1 unlabeled cells. Scale bars:
B, 100 µm; F, 25 µm.
|
|
CNTF stimulation changes mRNA and protein expression levels
of genes known to be involved in the Notch1 signaling pathway
The bHLH genes Hes1 and Hes5 are
known mediators of Notch signaling (Kageyama and Ohtsuka, 1999) and may
be involved in NSC or progenitor cell maintenance (Nakamura et al.,
2000, Ohtsuka et al., 2001). In addition, HES1 can directly
downregulate Mash1 expression, a gene whose expression is an
initial step in the NSC to progenitor cell transition (Chen et al.,
1997; Torii et al., 1999). We thus expected that gp130-mediated
signaling, initiated by CNTF, would increase the expression of
Hes1 and Hes5, with a concomitant decrease in
Mash1 expression. Surprisingly, 3 DIV P1 neurospheres
generated in EGF+CNTF appeared to show a downregulation in
Hes1 expression, although this did not achieve statistical significance (Fig. 9A)
(p > 0.05; n = 3).
Hes5 expression was significantly reduced in 3 DIV EGF+CNTF
P1 neurospheres compared with the equivalent neurospheres generated in
EGF alone (Fig. 9A) (p < 0.05;
n = 3). We also confirmed that there were no transient increases in Hes1 or Hes5 expression at 2 or 6 hr
after plating in EGF+CNTF compared with EGF alone (data not shown).
However, as expected, CNTF markedly decreased Mash1
expression in 3 DIV EGF+CNTF P1 neurospheres compared with EGF controls
(Fig. 9B) (p < 0.05;
n = 3). MASH1 has been reported to downregulate its own
mRNA expression (Meredith and Johnson, 2000); therefore we examined
MASH1 protein expression after CNTF treatment. We found that CNTF
treatment significantly decreases MASH1 expression in 3 DIV
neurospheres (Fig. 9B) (p < 0.05;
n = 3).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 9.
CNTF treatment changes the expression of genes
regulated by NOTCH1 signaling. A-C, Primary
neurospheres derived from the E14 striatum were grown in the presence
of EGF for 7 DIV, dissociated, and then cultured (5 × 104 cells/ml) in either EGF or EGF+CNTF. The cells
were then harvested for total RNA or protein at 3 DIV and processed for
RT-PCR Southern or Western blot analyses as described in Materials and
Methods. A, Constitutive CNTF treatment significantly
decreases Hes5 expression, whereas Hes1
expression in CNTF-treated P1 neurospheres does not differ
significantly from 3 DIV EGF-derived P1 neurospheres. B,
Mash1 expression, mRNA, and protein are significantly
reduced in P1 neurospheres cultured for 3 DIV in the presence of
EGF+CNTF compared with EGF alone. C,
Delta3 expression is reduced in 3 DIV EGF+CNTF P1
neurosphere cultures compared with EGF cultures. *p < 0.05 versus EGF; t test (A-C;
n = 3).
|
|
Given the decrease in MASH1 expression and because it is a
known transcriptional activator of Notch ligand expression (Casarosa et
al., 1999), we examined Delta/Serrate gene expression in EGF-generated neurospheres. RT-PCR analysis revealed that Delta1 and
-3 and Jagged1 and -2 were expressed
in EGF-generated P1 neurospheres (data not shown). Furthermore, we
found that the expression of Delta3 decreased (Fig.
9C) (p < 0.05, n = 3) in 3 DIV P1 neurospheres cultured in EGF+CNTF, compared with EGF
alone. The decrease in Delta3 expression is consistent with
the observed decrease in Mash1 mRNA and protein expression
in EGF+CNTF-treated P1 neurospheres and with our observation that
EGF+CNTF treatment of P1 neurospheres increases Notch1 expression.
| |
Discussion |
This report demonstrates, for the first time, a link
between gp130 and NOTCH1 signaling pathways in the regulation of NSC maintenance. Our results show a requirement for NOTCH1 signaling in the
maintenance and generation of NSCs. We find that gp130 signaling
specifically increases NOTCH1 expression in EGF-responsive NSCs
in vitro and in vivo. Furthermore, we find that
the increase in NOTCH1 expression is followed by an increase in the
ligand-mediated cleavage of NOTCH1-PF1 into NOTCH1-PF2 and further into
NOTCH1-PF3. Our observed decreases in Mash1 and
Delta3 are consistent with activation of gp130 leading to an
increase in NOTCH1 signaling. However, our results and a careful
reading of related studies (see below) suggest that Hes1 and
Hes5 may not be the critical components of NOTCH1 signaling
that are involved in NSC maintenance.
Glycoprotein130 activation regulates NOTCH1signaling, which is
required for the maintenance ofEGF-responsive NSCs
Recent evidence suggests that the phenotypic response to
Notch signaling in developing forebrain precursors is variable and dependent on temporal and spatial cues (Chambers et al., 2001). Moreover, it appears that Notch signaling is unable to regulate the
maintenance of neural crest stem cells (Morrison et al., 2000). Therefore, we first tested whether NOTCH1 expression and signaling play
a role in EGF-responsive NSC maintenance. Antisense to
Notch1 reduced its expression and consistently decreased
secondary neurosphere production. Furthermore, culture of neurospheres
in the presence of a -secretase inhibitor, known to reduce
production of activated NOTCH1 (De Strooper et al., 1999), also reduced
the production of secondary neurospheres (Fig. 2I).
Our results concur with the observations of Hitoshi and colleagues
(2002), implicating NOTCH1 signaling in the maintenance of
EGF-responsive NSCs. However, in contrast to their findings, we
demonstrate that blocking the processing of NOTCH1-PF2 to NOTCH1-PF3,
using a -secretase inhibitor, also inhibited NSCs from proliferating
in response to EGF. This suggests that NOTCH1 activation is required
for expansion and generation of EGF-responsive NSCs. Similarly, FGFs
require an intact Notch signaling pathway to prevent the
differentiation of E10.5 neuroepithelial progenitors cells into neurons
(Faux et al., 2001). The conclusion by Hitoshi et al. (2002) that Notch signaling is not required for the generation of NSCs is based on their
observation that
RBP-J
/
ES cells could give rise to the proliferation of primitive NSCs. However, the lineage relationship between these ES cell-derived NSCs
and in vivo generated NSCs is unclear, as is the role of ES
cell-derived NSCs in neurogenesis. Indeed, the fact that
NSCs could not be isolatedfrom
RBP-J/
or
presenilin1//presenilin2+/
embryos concurs with our data that Notch signaling is required for the
generation of NSCs.
We then tested the hypothesis that gp130-mediated signaling
regulates NOTCH1 signaling in NSCs. Our results demonstrate that CNTF specifically induces Notch1 mRNA and protein expression
(no change in Notch3 expression) in NSCs. With regards to
Notch1, our data are consistent with that of Faux et al.
(2001), who reported that LIF and members of the TGF family could
increase the expression of Notch1 in E10.5 neuroepithelial
progenitor cells, but inconsistent in the regulation of
Notch3, which was also upregulated by LIF and other factors
in their system. Although some of these discrepancies may be
attributable to the tissue or ontogenetic origin of the precursors,
Faux and colleagues (2001) did not explore how growth factors regulated
Notch1 signaling or its relevance to NSC function. In vivo,
we found that CNTF, in the presence of EGF, upregulates the number of
NOTCH1-expressing cells [as we reported, it increases NSC numbers
(Shimazaki et al., 2001)] in the adult forebrain periventricular area, which is the location of adult NSCs. The ability of sIL6R+IL6 to
enhance NOTCH1 and NSC numbers in
LIFR/
neurospheres demonstrates that gp130 activation mediates the increases
in NOTCH1 expression and NSC maintenance. These data concur with the
recent findings of Hatta et al. (2002) that signaling by gp130 keeps
embryonic precursors in the stem cell state and suggests that this
effect may be mediated by NOTCH1.
The observations that CNTF enhances NOTCH1-PF1 expression rapidly and
in the absence of cell-cell contact suggests a direct action on NSCs
that does not involve lateral inhibition. This further suggests that
the decrease of Delta3 that we observed is a
result and not a cause of increased NOTCH1 signaling. On the other
hand, we demonstrate that cell-cell contact is required for CNTF to
increase NOTCH1-PF2 and NSC self-renewal (Fig.
4B,C).
EGF and gp130 signaling pathways cooperatively regulate NOTCH1
signaling in NSCs and may establish the pattern of cell
contact-mediated signaling during development
The results of this study suggest that the gp130-mediated increase
in NOTCH1 signaling is context dependent. CNTF only induced an increase
in NOTCH1 expression in the presence of either EGF or FGF2. Figures
4A-F and 7B demonstrate that EGF alone
can increase NOTCH1 expression in vitro. Both EGF and CNTF
can phosphorylate tyrosine residues on STAT3, which is necessary
for the dimerization of STAT3 and its translocation to the
nucleus (for review, see Akira, 1999; Turnley and Bartlett, 2000).
Additionally, it has been reported that MAP kinase can phosphorylate
dimerized STAT proteins on serine residues, which appears
to be necessary for STAT-dependent transcriptional activation (Akira,
1999). Although the upstream 5' sequence of mouse Notch1 is
unavailable to us, we examined the genomic sequence upstream of
Drosophila Notch for the presence of STAT binding sites. The
existence of two putative STAT-element binding sites, 5'-TTCNNNGAA in
Drosophila (Kwon et al., 2000) at 954:962 and
1035:1043 with respect to the Notch start codon
(designated as 0), is highly suggestive that gp130-mediated JAK/STAT signaling may directly regulate the transcription of Notch1 in Drosophila and in the mouse. Thus, it
is plausible that EGF and CNTF signaling may cooperatively activate the
dimerization, translocation, and activation of STAT3 proteins, which
may in turn act to promote Notch1 expression.
EGF- and gp130-mediated signaling may also cooperate to
establish the pattern of NOTCH1 signaling within the developing CNS. For example, cells within the ventricular zone, exposed to high levels
of CNTF/LIF and EGF signaling, would express high levels of NOTCH1 and
through lateral inhibition "determine" how cell-cell contact-mediated signaling would allow distinction/separation of the
adjacent progenitor cell pool in the subventricular/mantle zones. Thus
cells further removed from EGF/CNTF would become progenitor cells,
limited in their capacity to self-renew and more able to express genes,
such as Mash1, involved in the determination of restricted
neural progenitor cells. Indeed, EGF+CNTF decreased Mash1 mRNA and protein expression in NSC cultures, as this
model would predict. A similar concept was suggested previously by
Price et al. (1997), with respect to Notch and EGFR signaling in the establishment or maintenance of posterior follicle cell fates in
Drosophila. They provide evidence suggesting that EGFR
signaling influences NOTCH signaling in posterior follicle cells and
the establishment of the expression levels of DELTA ligand, which would, in turn, be maintained by lateral inhibition. Thus, as reported
in other systems, our results support the contention that cell
contact-mediated signaling and non-cell contact-mediated epigenetic
signaling pathways are intimately linked in the establishment of neural
patterning and development.
Mediation of NSC maintenance and proliferation by NOTCH1 signaling
may be independent of Hes1 and Hes5
In this study, we found that neurospheres
cultured for 3 DIV in EGF+CNTF demonstrate a fivefold increase in
NOTCH1 expression compared with EGF controls. This increase in NOTCH1
expression is concomitant with a significant decrease in
Hes5 expression and a trend toward a decrease (did not
achieve statistical significance) in Hes1 expression. Given
the decrease in the progenitor determination gene Mash1
(Fig. 9B), a gene whose transcription is repressed by
Hes1 (Chen et al., 1997), it is surprising that there was no increase in Hes gene expression in the CNTF
treated-cultures. However, these observations are not unlike those made
by Shawber et al. (1996) whereby NOTCH1 activation by JAGGED1, which
kept C2C12 myoblasts from differentiating, did not result in the
upregulation of Hes1. Stable transfection of C2C12 myoblasts
with Hes1 was also unable to inhibit their differentiation.
Additionally, Furukawa et al. (2000) reported that overexpression of
activated Notch1 in the retina increased clone size, whereas
overexpression of Hes1 did not. Finally, there was no
decrease found in the expression of Hes1 in
presenilin1/
brains (Handler et al., 2000) or in
RBP-J/
ES cell sphere colonies (Hitoshi et al., 2002) where there were decreases in the self-renewal of isolated NSCs. These studies are
consistent with the notion that Hes1 is not necessarily
involved in promoting an undifferentiated state.
The suggestion that Hes genes do not function primarily in
NSC maintenance is in apparent contrast to the studies by Nakamura et
al. (2000) and Ohtsuka et al. (2001), where neurospheres could be
generated from embryos mutant for Hes1 and/or
Hes5, yet a role for these factors in NSC maintenance was
suggested. In both studies, single or double mutant neurospheres were
on average smaller; however, the double mutant primary neurospheres
could still produce P1 neurospheres (demonstrating self-renewal). In
fact, when neurospheres were normalized for total cell number,
Ohtsuka et al. (2001) found that single mutants did not show a reduced
number of secondary neurospheres. In our previous study (Shimazaki et
al., 2001) and in the current report, reduction in self-renewal is
defined as a reduced number of P2 neurospheres from single equivalent
sized P1 neuropheres, or as a population normalized for cell number. The observations of smaller primary or secondary neurospheres, reported
by Nakamura et al. (2000) and Ohtsuka et al. (2001), could be as
readily interpreted as Hes1 and Hes5 functioning
primarily in the maintenance of neural progenitor cells, indeed as
suggested by Nakamura et al. (2000), with regard to Hes1.
Furthermore, the observation that no neurospheres could be obtained
from
presenilin1//presenilin2+/
mice (Hitoshi et al., 2002), in contrast to
Hes1/Hes5 double mutants, strongly supports the
contention that other factors can mediate NOTCH1 signaling actions on
NSC self-renewal. Kondo and Raff (2000) reported that both
Mash1 and Hes5 are expressed in oligodendrocyte
progenitors and may mediate their differentiation. Additionally,
Hes5 appears to function in later progenitors of the
olfactory neuroepithelium (Cau et al., 2000). Therefore, the marked
decrease in Hes5 expression observed in 3 DIV EGF+CNTF compared with EGF cultures may be the result of an increase in NSCs at
the expense of progenitor cells with a limited self-renewal capacity,
consistent with our previous findings (Shimazaki et al., 2001) that
CNTF supports the maintenance of NSCs by suppressing their restriction
to glial progenitors.
Considering that two new Hes genes, Hes6 and
Hes7 (Bae et al., 2000; Bessho et al., 2001), have been
discovered recently, it is plausible that an as yet unidentified
Hes gene mediates the increase in NOTCH1 signaling
stimulated by CNTF. Given their observation of neurosphere formation in
the Hes1/Hes5 double mutants, Ohtsuka and colleagues (2001)
suggest that a splice variant of Hes3 (Hes3b) may
contribute to embryonal NSC maintenance. It is also possible that a
SuH/RBP-J-independent pathway, which
may not require Hes, mediates the CNTF-induced increase in
NOTCH1 signaling (Shawber et al., 1996; Matsuno et al., 1997;
Ordentlich et al., 1998). Future studies of gp130 regulation of
Hes genes will likely serve to identify the family member
that mediates NOTCH1 regulation of NSC maintenance and
self-renewal.
| |
FOOTNOTES |
Received June 24, 2002; revised Dec. 6, 2002; accepted Dec. 9, 2002.
This work was supported by the Canadian Institutes of Health Research.
C.G. is supported by a studentship from the Multiple Sclerosis
Foundation of Canada. S.W. is an Alberta Heritage Foundation for
Medical Research Scientist. We thank Dorothea Livingstone for excellent
technical assistance and Dr. Carol Schuurmans for critical
review of an earlier version of this manuscript.
Correspondence should be addressed to Samuel Weiss, Genes & Development
Research Group, Department of Cell Biology and Anatomy, University of
Calgary, Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1. E-mail:
weiss{at}ucalgary.ca.
T. Shimazaki's present address: Department of Physiology, Keio
University School of Medicine, 35 Shinanomachi, Shinjyuku-ku Tokyo,
160-8582 Japan.
| |
References |
-
Akira S
(1999)
Functional roles of STAT family proteins: lessons from knockout mice.
Stem Cells
17:138-146[Abstract/Free Full Text].
-
Alvarez-Buylla A,
Herrera DG,
Wichterle H
(2000)
The subventricular zone: source of neuronal precursors for brain repair.
Prog Brain Res
127:1-11[Medline].
-
Alvarez-Buylla A,
Garcia-Verdugo JM,
Tramontin AD
(2001)
A unified hypothesis on the lineage of neural stem cells.
Nat Rev Neurosci
2:287-293[ISI][Medline].
-
Artavanis-Tsakonas S,
Rand MD,
Lake RJ
(1999)
Notch signaling: cell fate control and signal integration in development.
Science
284:770-776[Abstract/Free Full Text].
-
Austin CP,
Feldman DE,
Ida Jr JA,
Cepko CL
(1995)
Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch.
Development
121:3637-3650[Abstract].
-
Bae S-K,
Bessho Y,
Hojo M,
Kageyama R
(2000)
The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation.
Development
127:2933-2943[Abstract].
-
Bessho Y,
Miyoshi G,
Sakata R,
Kageyama R
(2001)
Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm.
Genes Cells
6:175-185[Abstract].
-
Brou C,
Logeat F,
Gupta N,
Bessia C,
Lebail O,
Doedens JR,
Cumano A,
Roux P,
Black RA,
Israël A
(2000)
A novel proteolytic cleavage involved in notch signaling: the role of the disintegrin-metalloprotease TACE.
Mol Cell
5:207-216[ISI][Medline].
-
Carpenter MK,
Cui X,
Hu Z,
Jackson J,
Sherman S,
Seiger A,
Wahlberg LU
(1999)
In vitro expansion of a multipotent population of human neural progenitor cells.
Exp Neurol
158:265-278[ISI][Medline].
-
Casarosa S,
Fode C,
Guillemot F
(1999)
Mash1 regulates neurogenesis in the ventral telencephalon.
Development
126:525-534[Abstract].
-
Cau E,
Gradwohl G,
Casarosa S,
Kageyama R,
Guillemot F
(2000)
Hes genes regulate sequential stages of neurogenesis in the olfactory epithelium.
Development
127:2323-2332[Abstract].
-
Chambers CB,
Peng Y,
Nguyen H,
Gaiano N,
Fishell G,
Nye JS
(2001)
Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors.
Development
128:689-702[Abstract].
-
Chen H,
Thiagalingam A,
Chopra H,
Borges MW,
Feder JN,
Nelkin BD,
Baylin SB,
Ball DW
(1997)
Conservation of the Drosophila lateral inhibition pathway in human lung cancer: a hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression.
Proc Natl Acad Sci USA
94:5355-5360[Abstract/Free Full Text].
-
Conover JC,
Ip NY,
Poueymirou WT,
Bates B,
Goldfarb MP,
DeChiara TM,
Yancopoulos GD
(1993)
Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells.
Development
119:559-565[Abstract]
.gif?ad=17923&adview=true)
TOP
ABSTRACT
Introduction
