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First published online 25 May 2006
doi: 10.1242/dev.02419
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1 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
2 Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu
University, Fukuoka, Japan.
3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan.
* Author for correspondence (e-mail: ygotoh{at}iam.u-tokyo.ac.jp)
Accepted 27 April 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretase
inhibitor abrogated the enhancement of neurosphere formation by STAT3. Our
results reveal a previously unrecognized mechanism of interaction between the
JAK-STAT3 and DLL1-Notch signaling pathways, as well as a pivotal role for
this interaction in maintenance of NPCs during early neocortical
development.
Key words: STAT3, Neural precursor cell, Neocortex, FGF2, Notch, DLL, Non-cell-autonomous
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Davis and Temple,
1994; Gage, 2000;
McKay, 1997). In the early
stages of brain development, NPCs primarily increase in number. They
proliferate and generate neurons and glial cells in a sequential manner during
mid-to-late gestation and postnatal development, respectively
(McConnell, 1995;
Qian et al., 2000). The final
number of neurons in each region of the brain is determined by various
factors, including the number of times that NPCs divide in the
undifferentiated state before undergoing the neuronal differentiation that
ultimately leads to cell cycle arrest. The maintenance of NPCs in the
undifferentiated state is thus strictly regulated during brain development,
although the underlying mechanisms are not fully understood. Such regulation
is also an important determinant of the pool size of NPCs in the adult
brain.
Maintenance of NPCs is supported by two types of extracellular cues: Notch
ligands and secreted growth factors. Notch is a transmembrane receptor that is
activated on cell-cell contact by its ligands delta-like and jagged expressed
in neighboring cells (Artavanis-Tsakonas et
al., 1999; Lai,
2004; Yoon and Gaiano,
2005). After such ligand binding, the intracellular domain of
Notch is released as a result of cleavage by the presenilin (PS)-containing
-secretase complex and translocates to the nucleus, where it activates
the transcription factor RBP-J. Activation of Notch signaling results in the
expression of target genes such as those for HES1 and HES5
(Artavanis-Tsakonas et al.,
1999; Honjo, 1996;
Kageyama and Nakanishi, 1997;
Selkoe and Kopan, 2003;
Yoon and Gaiano, 2005), both
of which inhibit the activity and expression of proneural basic
helix-loop-helix (bHLH) proteins. Misexpression of activated NOTCH1 or of HES1
or HES5 in the developing mouse forebrain inhibits neuronal differentiation
and results in expansion of the NPC pool
(Gaiano et al., 2000;
Ohtsuka et al., 2001), whereas
mice lacking PS1, PS2, NOTCH1, RBP-J, or both HES1 and HES5 manifest premature
neurogenesis accompanied by a loss of NPCs
(Cau et al., 2000;
de la Pompa et al., 1997;
Donoviel et al., 1999;
Handler et al., 2000;
Lutolf et al., 2002;
Ohtsuka et al., 1999).
The abundance of NPCs can be estimated in vitro by culture of neural cells
in suspension at a low density and determination of the efficiency of
formation of `neurospheres' that contain cells capable both of generating new
neurosphere colonies on dissociation and of differentiating into neurons and
glia (Reynolds and Weiss,
1996; Tropepe et al.,
1999). Inactivation of the Notch signaling pathway by treatment
with a -secretase inhibitor or by disruption of NOTCH1, PS1, RBP-J, or
both HES1 and HES5 genes impaired the ability of mouse neuroepithelial cells
(or embryonic stem cell-derived neural cells) to form passageable neurospheres
(Chojnacki et al., 2003;
Hitoshi et al., 2002;
Ohtsuka et al., 2001), further
supporting the notion that the Notch pathway is essential for NPC
maintenance.
Various secreted growth factors are also thought to contribute to the
maintenance of NPCs. The activity of fibroblast growth factor 2 (FGF2) in this
regard has been widely taken advantage of to isolate and expand NPCs in a
variety of in-vitro systems (Gritti et
al., 1999; Kilpatrick and
Bartlett, 1993; Mayer-Proschel
et al., 1997; Reynolds et al.,
1992; Reynolds and Weiss,
1992; Reynolds and Weiss,
1996; Tropepe et al.,
1999; Vescovi et al.,
1993). FGF2-responsive NPCs first appear in the mouse neocortex as
early as embryonic day (E) 8.5, and an additional population of epidermal
growth factor (EGF)-responsive NPCs arises from these FGF2-responsive cells
between E11 and 13 (Burrows et al.,
1997; Mayer-Proschel et al.,
1997; Tropepe et al.,
1999). Mice lacking FGF2 exhibit a reduced density and number of
neurons in the neocortex (Dono et al.,
1998; Ortega et al.,
1998; Raballo et al.,
2000), whereas microinjection of FGF2 into the cerebral ventricles
at early stages of mouse embryogenesis increased the number of rounds of
division of NPCs, resulting in an increased number of neurons
(Vaccarino et al., 1999). FGF2
signaling thus appears to contribute to NPC maintenance during embryogenesis,
although the mechanism of this effect remains largely unknown.
We have now found that STAT3 (signal transducer and activator of
transcription 3) is responsible for the maintenance of mouse embryonic
neocortical NPCs by FGF2. STAT3 is a transcription factor that is activated as
a result of phosphorylation on Tyr705 by tyrosine kinases such as
Janus kinase (JAK). Such phosphorylation induces dimerization of STAT3 and its
translocation to the nucleus, where it modulates transcription through
interaction with specific DNA elements
(Levy and Darnell, 2002).
STAT3 promotes astrocytic differentiation in the postnatal mouse neocortex
(Bonni et al., 1997;
Rajan and McKay, 1998), but
its roles in the early embryonic neocortex have been unclear. We found that
STAT3 is expressed specifically in NPCs at early stages of neocortical
development, and that deletion of the STAT3 gene resulted in premature
neuronal differentiation of neighboring cells. We also show that Delta-like1
(DLL1) is an essential component of the STAT3-mediated signaling pathway
responsible for NPC maintenance, demonstrating a nexus between the Delta-Notch
and JAK-STAT3 pathways that regulates the fate of NPCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All pharmacological signaling pathway inhibitors were obtained from Calbiochem (AG490, LY294002, U0126, L685,458).
Primary culture
Neuroepithelial cells were isolated as described previously
(Hirabayashi et al., 2004).
The cells were cultured in medium comprising 1:1 (v/v) mixture of Dulbecco's
modified Eagle's medium and F12 medium (Gibco) supplemented with B27
(Invitrogen) with or without FGF2 (20 ng/ml, R&D) or EGF (20 ng/ml,
Upstate Biotechnology).
Neuroepithelial cell culture
The cells were transferred to noncoated 100-mm dishes for 90 minutes to
remove non-neural cells, and the remaining floating cells were plated on
poly-D-lysine-coated dishes to yield a neuroepithelial cell culture, which was
maintained in the presence of FGF2 (20 ng/ml). Infection of these cells with a
retrovirus encoding GFP (pMX-GFP) and subsequent immunofluorescence analysis
revealed that >90% of the nonfibroblastic GFP-positive cells expressed
nestin (fibroblast-like cells were excluded from the analysis on the basis of
their distinct morphology).
NPC culture
To obtain an NPC-enriched population, we plated the dissociated
neuroepithelium directly on noncoated 100-mm dishes in culture medium
containing FGF2 (20 ng/ml) and EGF (20 ng/ml) and cultured the cells for 3
days. The resulting neurospheres were then dissociated and plated on
poly-D-lysine-coated dishes in culture medium containing FGF2 (20 ng/ml) to
yield an NPC culture.
Immunoblot analysis
Immunoblot analysis was performed as described
(Kamakura et al., 2004).
Primary antibodies included rabbit polyclonal antibodies to STAT3 (1:1000
dilution, Upstate Biotechnology) or to the Tyr705-phosphorylated
form of STAT3 (1:200, Cell Signaling), with mouse monoclonal antibodies to
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Chemicon), or with
goat polyclonal antibodies to DLL1 (1:100, C-20, Santa Cruz
Biotechnology).
Neurosphere assay
Completely dissociated primary neuroepithelial cells were suspended in
culture medium containing FGF2 (20 ng/ml) at a density of 5000 cells/ml, and
200 µl of the cell suspension were transferred to each well of a 96-well
plate. The number of primary neurospheres was counted after culture for 7
days. About 5% of neuroepithelial cells prepared from E12.5 mouse neocortex
generated neurospheres in the presence of FGF2, whereas no neurospheres formed
in the absence of FGF2. About 90% of the primary neurospheres formed in the
presence of FGF2 were multipotent, in that they were able to differentiate
into both neurons and glia when plated on poly-D-lysine-coated dishes in the
absence of FGF2 (data not shown).
For assay of the formation of secondary neurospheres, primary neurospheres formed at a cell density of 2.5x105 cells/ml in 6-well plates and culture medium containing FGF2 (20 ng/ml) were collected and dissociated. Portions (200 µl) of cell suspension (5000 cells/ml) were then transferred to each well of a 96-well plate in culture medium containing FGF2 (20 ng/ml) and EGF (20 ng/ml). The number of secondary neurospheres was counted after culture for 7 days.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline
(PBS) and incubated with primary antibodies for 2 hours. Primary antibodies
included rabbit anti-GFP (1:500 dilution, Medical and Biological
Laboratories), mouse anti-GFP (1:500, Chemicon), mouse anti-CD8 (1:200,
PharMingen), mouse anti-nestin (1:200, Becton Dickinson), mouse
anti-ßIII-tubulin (TuJ1) (1:500, Babco), rabbit anti-GFAP (1:2000, DAKO),
and mouse anti-BrdU (1:50, BD). Immune complexes were detected with Alexa
Fluor 488- or Alexa Fluor 594-conjugated goat secondary antibodies (Molecular
Probes).
Immunohistochemistry
Immunohistochemistry was performed as described
(Hirabayashi et al., 2004).
For staining with anti-DLL1, antigen retrieval was accomplished by autoclave
treatment of sections for 15 minutes at 105°C in 0.01 mol/l sodium citrate
buffer (pH 6.0) and subsequent washing with Tris-buffered saline containing
0.05% Tween 20 (TBS-T). Primary antibodies included rabbit anti-STAT3 (1:100
dilution, Sigma), rabbit anti-GFP (1:500), mouse anti-GFP (1:500), mouse
anti-nestin (1:200), mouse anti-ßIII-tubulin (1:1000), rabbit anti-SOX2
(1:500, Chemicon), mouse anti-MAP2 (1:500, HM-2, SIGMA), rabbit anti-Musashi
(1:500, Chemicon), and rabbit anti-DLL1 (1:100, H-265, Santa Cruz
Biotechnology). The sections were washed in TBS-T and then incubated for 1
hour at room temperature with donkey secondary antibodies conjugated with
Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Molecular
Probes), or with biotin-conjugated secondary antibodies before exposure to
reagents first of an ABC kit (Vector Laboratories) and then of a TSA kit
(Molecular Probes).
In situ hybridization
In-situ hybridization on frozen sections was performed basically as
described previously (Nomura and Osumi,
2004). Probe for mouse STAT3 was prepared from FANTOM2 clone set
(Okazaki et al., 2002).
In utero electroporation
Introduction of plasmid DNA into neuroepithelial cells of mouse embryos in
utero was performed as described (Tabata
and Nakajima, 2001). Plasmid DNA (pMX-GFP or pMX-Cre-GFP, at 1
mg/ml) was injected into the lateral ventricle of each littermate at E14.5.
Two days after electroporation, the embryos were harvested and the brains were
examined immunohistochemically.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed as described previously
(Hirabayashi et al., 2004).
Mouse anti-STAT3 (C-20) or control immunoglobulin G (Santa Cruz Biotechnology)
was used. The sense and antisense primers for amplification of fragments of
the DLL1 locus were 5'-TGTGACATCTACAATGGCACTA-3' and
5'-AGCCCAAAGGTAACATAAATCTC-3' (nucleotides -6777 to -6638,
relative to the transcription start site),
5'-TGGTTTCCACTTCCATCTTCTAA-3' and
5'-TCTGAAGCTGCTCTCTGTTG-3' (nucleotides -2819 to -2639),
5'-CTTTCTCACCTCAGCCTT-3' and 5'-ATGACCCAGCCTGCCTA-3'
(nucleotides -2370 to -2211), 5'-ATTACCATACAGCTGAGCG-3' and
5'-TTCACTGGTGTCCGGTC-3' (nucleotides -1486 to -1323),
5'-ACTCTCCGAGGCTCAATC-3' and 5'-CCTGGCAGCCTCTTTCTA-3'
(nucleotides -1118 to -937), 5'-AGCGTGCCTAGGGTCTTCGGG-3' and
5'-CGCTCATTTACATTCCTGCAA-3' (nucleotides -760 to -644), and
5'-GGTGTGCTAGAATACCATTATGAA-3' and
5'-ATGGAGACAACCTGGGTATC-3' (nucleotides +4572 to +4697),
respectively.
RT-PCR
Total RNA was isolated from infected NPC cultures with the use of the
TRIzol reagent (Invitrogen) and was subjected to RT with an
oligo(dT)12-18 primer (Invitrogen) and ReverTra Ace reverse
transcriptase (Toyobo). The resulting cDNA was subjected to real-time PCR in a
Roche LightCycler with SYBR-green Realtime PCR Master Mix. The abundance of
target mRNAs was normalized relative to that of GAPDH mRNA. The sense and
antisense primers, respectively, were as follows: GAPDH,
5'-TGGGTGTGAACCACGAG-3' and
5'-AAGTTGTCATGGATGACCTT-3'; NOTCH1,
5'-TCAGAGGCCACATAGCTTC-3' and
5'-TAAATGCCTCTGGAATGTGGGTGAT-3'; DLL1,
5'-TGTTCTAACGGTGCCAAG-3' and 5'-GGCACAGTCATCCACATT-3';
and GFAP, 5'-TGGCTCGTGTGGATTTGGAG-3' and
5'-TGTACAGGAATGGTGATGCG-3'.
RNA interference
Cells were depleted of DLL1 by RNA interference (RNAi) with the use of an
RNAi-Ready pSIREN vector (BD Biosciences). The pSIREN-siLuc (control siRNA)
and pSIREN-siDLL1 (DLL1 siRNA) retroviral constructs were generated in
accordance with the manufacturers' instructions (BD Biosciences and Clontech).
The target sequence for the DLL1 siRNA was
5'-GTAGATGAGTGTGCTCCTAGC-3'.
Statistical analysis
Quantitative data are presented as means ± s.e.m. from
representative experiments that were repeated a total of more than three times
with similar results. Values were compared with the unpaired Student's
t test. A P-value of <0.05 was considered statistically
significant.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hitoshi et al., 2002;
Molofsky et al., 2003;
Weiss et al., 1996). In this
assay, neuroepithelial cells are dissociated and cultured in suspension at a
low density (<15,000 cells/ml) so that most of the resulting cell
aggregates (neurospheres) are derived from single cells
(Hulspas et al., 1997). We
first examined whether signaling pathways mediated by the mitogen-activated
protein kinase ERK and its upstream regulator MEK, by phosphatidylinositol
3-kinase (PI3K), or by JAK and STAT contribute to the FGF2-dependent formation
of neurospheres with the use of pharmacological inhibitors. Neuroepithelial
cells prepared from E12.5 mice were dissociated and cultured with FGF2 in the
absence or presence of either the MEK inhibitor U0126 (10 µmol/l), the PI3K
inhibitor LY294002 (20 µmol/l), or the JAK2 inhibitor AG490 (10 µmol/l).
Treatment of neuroepithelial cells with these concentrations of U0126,
LY294002 or AG490 reduced the level of phosphorylation of ERK, Akt and STAT3,
respectively, observed in the presence of FGF2 (data not shown), confirming
that these reagents effectively blocked the corresponding signaling pathways.
Culture of the cells for 7 days with AG490 resulted in marked inhibition of
neurosphere formation, whereas LY294002 exhibited a smaller (but significant)
inhibitory effect and U0126 had no effect
(Fig. 1A). These results
suggest that JAK2 and PI3K contribute to FGF2-mediated formation of
neurospheres. To examine whether JAK2 and PI3K are involved in NPC maintenance, we next exposed primary neurospheres generated by incubation of neuroepithelial cells in the presence of FGF2 to the inhibitors during day 3 of the culture. The neurospheres were then dissociated and replated in the presence of FGF2 and EGF to allow the formation of secondary neurospheres in the absence of inhibitors (Fig. 1B). In this assay, >80% of the resulting secondary neurospheres generated neurons, astrocytes and oligodendrocytes when cultured under differentiation-promoting conditions (data not shown), indicating that most of the neurosphere-forming cells were multipotent NPCs. Exposure of the primary neurospheres to AG490, or to U0126 and LY294002 together, or to LY294002 alone resulted in a significant decrease in the number of secondary neurospheres (Fig. 1B; see Fig. S1A in the supplementary material). The proliferation rate of overall neuroepithelial cells was slightly inhibited by AG490, whereas the survival rate was not much affected (see Fig. S1C,D in the supplementary material). However, AG490 increased the percentage of neuron-only clones in a clonal assay (see Fig. S1B in the supplementary material). These results together suggest that the activity of JAK2 is required for FGF2-dependent NPC maintenance (which includes promotion of survival/proliferation and inhibition of differentiation of NPCs).
STAT3 is required for NPC maintenance
JAK2 activates members of the STAT family of transcription factors
(Levy and Darnell, 2002).
Among the members of this family, STAT3 is abundant and expressed in the brain
(De-Fraja et al., 1998;
Yan et al., 2004). We found
that the expression of STAT3 in the forebrain of E13.5 and 16.5 mouse embryos
overlapped in part with that of the NPC marker nestin
(Fig. 2A-F,H,J). Nestin was
exclusively expressed in cells in the VZ and in radial fibers, whereas STAT3
was expressed in these nestin-positive cells as well as in cells in the
subventricular zone (SVZ) and the apical side of the intermediate zone (IMZ)
(Fig. 2K-R). In-situ
hybridization analysis revealed a distribution of STAT3 mRNA similar to that
of the encoded protein (Fig.
2S). By contrast, the expression of STAT3 was not detected in
cells positive for the neuronal marker TuJ1 (ßIII-tubulin)
(Fig. 2G,I), suggesting that
STAT3 is abundant in NPCs and downregulated after neuronal
differentiation.
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), in which both Stat3 alleles are flanked by
LoxP sites. Infection of the cells with a retrovirus encoding the Cre
recombinase (pMX-Cre-GFP) resulted in an 80% decrease in the abundance of
STAT3 within 3 days compared with that apparent in cells infected with a
control retrovirus (Fig. 3B).
The FGF2-dependent formation of primary neurospheres by the STAT3-depleted
cells was inhibited by 47% (±8% s.e.m.) compared with that observed
with the control cells (Fig.
3C). Introduction of Cre recombinase did not markedly reduce the
number of secondary neurospheres, most probably because of a selective
expansion of STAT3-remaining neurospheres. Expression of a dominant negative
form of mouse STAT3 also reduced by 47% (±14% s.e.m.) the number of
primary neurospheres formed by wild-type neuroepithelial cells expressing
control vector. Moreover, expression of a constitutively active form of mouse
STAT3 (STAT3-C, which harbors cysteine mutations at Ala661 and
Asn663) (Bromberg et al.,
1999) increased in the number of secondary neurospheres formed by
wild-type neuroepithelial cells (Fig.
3D). The proliferation and survival rates of these cells, however,
were not much affected by the manipulation of STAT3 (see
Fig. 3E,F,H,I). These results
suggest that STAT3, in addition to JAK2, is important for the maintenance of
NPCs in vitro.
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; Takizawa et al.,
2001). These results indicated that STAT3 inhibits neuronal
differentiation of NPCs in this culture system.
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STAT3 inhibits neurogenesis in a non-cell-autonomous manner
Reduced SOX2 expression in GFP-positive cells at the VZ in
Stat3-deleted experiments suggests that STAT3 is necessary for the
maintenance of NPC in a cell-autonomous manner. Detailed examination of the
results of the in-vivo electroporation experiment, however, revealed that
deletion of Stat3 in NPCs (as reflected by GFP expression) also
resulted in ectopic neuronal differentiation in surrounding GFP-negative cells
(Fig. 4B, arrows). Indeed, a
large proportion [94% (±1.9% s.e.m.)] of the TuJ1-positive cells in the
STAT3-deleted VZ area were GFP-negative. This observation suggests that STAT3
is required for inhibition of neurogenesis in surrounding cells.
To verify whether STAT3 indeed inhibits neurogenesis in a non-cell-autonomous manner, we performed a series of co-culture experiments. A neuroepithelial cell culture prepared from the neocortex of E12.5 STAT3flox/flox mice was infected with a retrovirus encoding CD8 (pMX-CD8) or one encoding both CD8 and Cre recombinase (pMX-Cre-CD8). The infection efficiency was relatively high (>80% of NPCs were CD8-positive) in these experiments. These cells were then co-cultured with a small number (1% of the number of CD8-expressing cells) of cells from an NPC culture that had been infected with a retrovirus encoding GFP (pMX-GFP) (Fig. 5A). After incubation for 4 days, the proportion of GFP-positive NPC clones that expressed TuJ1 was markedly greater for co-cultures with neuroepithelial cells infected with pMX-Cre-CD8 (STAT3 depleted) than for those with neuroepithelial cells infected with pMX-CD8 (Fig. 5B). This result again suggests that STAT3 is required for inhibition of neuronal differentiation in surrounding cells.
We then co-cultured GFP-expressing NPCs with either control neuroepithelial cells or those expressing STAT3-C. A neuroepithelial cell culture prepared from wild-type mouse embryos was thus infected with pMX-CD8 or the corresponding vector encoding both STAT3-C and CD8 (pMX-STAT3-C-CD8). These cells were then co-cultured with a small number of GFP-expressing NPCs for 3 days. The proportion of neuron-only clones among GFP-positive clones was markedly smaller for co-cultures with neuroepithelial cells infected with pMX-STAT3-C-CD8 than for those with neuroepithelial cells infected with pMX-CD8 (Fig. 5C), further supporting the notion that STAT3 can inhibit neuronal differentiation of NPCs in a non-cell-autonomous manner.
|
).
Immunohistofluorescence analysis indeed showed that the expression of DLL1
overlapped with that of STAT3 in the mouse embryonic neocortex
(Fig. 6A-D), which prompted us
to examine the functional relation between STAT3 and DLL1.
|
;
Faux et al., 2001). Immunoblot
analysis showed that the amount of DLL1 protein in NPC cultures was also
reduced as a result of Stat3 deletion
(Fig. 6F). These results thus
suggest that STAT3 is required for DLL1 expression in the NPC culture, which
contains both NPCs and newborn neurons.
We next examined whether STAT3 directly associates with the promoter of
Dll1 in NPC cultures by ChIP analysis. Five putative consensus
sequences for STAT3 binding (Ehret et al.,
2001) were found to be located at nucleotide positions -665 to
-657, -1315 to -1307, -1393 to -1385, -2622 to -2614, and -2717 to -2709
relative to the transcription start site in the promoter region of the mouse
DLL1 gene (Fig. 6G), including
two sequences conserved among mouse and human (-1393 to -1385 and -665 to
-657). The region between nucleotides -1 to -4265 of the mouse DLL1 gene has
been shown to be sufficient to reproduce the normal pattern of Dll1
expression during early organogenesis
(Beckers et al., 2000).
Chromatin immunoprecipitation assay prepared from NPC lysates with anti-STAT3
revealed the presence of genomic DNA containing the three regions (-2819 to
-2639, -1486 to -1323, -760 to -644), which include the putative STAT3-binding
sequences within the Dll1 promoter region
(Fig. 6G). By contrast, PCR
analysis with primers targeted to other regions of Dll1 did not yield
a signal with anti-STAT3 immunoprecipitates greater than that obtained with
control immunoprecipitates (Fig.
6G). These results thus suggest that STAT3 might directly regulate
the transcription of Dll1.
Suppression of Notch signaling inhibits NPC maintenance by STAT3
Our observations indicated that STAT3 contributes to NPC maintenance and
promotes expression of DLL1. We therefore next examined whether DLL1
expression is necessary for STAT3-mediated NPC maintenance with the use of
DLL1-directed RNAi. Transfection of neuroepithelial cell cultures with a
vector for an siRNA specific for DLL1 mRNA resulted in an 
80% decrease in
the abundance of DLL1 protein (Fig.
7A). The potentiation of secondary neurosphere formation induced
by expression of STAT3-C in NPCs was prevented by RNAi-mediated depletion of
DLL1 (Fig. 7B). We also found
that treatment of neuroepithelial cells with the -secretase inhibitor
L685,458, which inhibits Notch receptor activation, blocked the stimulatory
effect of STAT3-C on secondary neurosphere formation
(Fig. 7C). Together, these
results suggest that DLL1 is an essential mediator of the action of STAT3 in
NPC maintenance.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The extracellular signals responsible for the activation of STAT3 in the
developing neocortex remain to be identified, although we used FGF2 to promote
maintenance of NPCs in vitro. Characterization of FGF2 knockout mice has
indicated that FGF2 partially contributes to the maintenance of neocortical
NPCs in vivo (Raballo et al.,
2000). EGF also promotes the expansion of NPCs, especially at the
late stage of neocortical development
(Reynolds and Weiss, 1996).
Activation of the EGF receptor results in an increase in the amount of STAT3
mRNA, which may account in part for NPC maintenance at this late stage
(Viti et al., 2003). In
addition to FGF2 and EGF, gp130-coupled signaling molecules such as LIF,
ciliary neurotrophic factor and interleukin-6 activate STAT3
(Cattaneo et al., 1999). LIF
and its receptor might also contribute to NPC maintenance by activating STAT3,
especially in the embryonic ganglionic eminences and in the adult SVZ, given
that deletion of the gene for LIFRß, the ß subunit of the LIF
receptor, reduced the capacity of cells from these regions to form
neurospheres (Gregg and Weiss,
2005; Shimazaki et al.,
2001), that the addition of LIF increased the number of
passageable neurospheres generated from ganglionic eminences
(Gregg and Weiss, 2005), and
that injection of LIF into the lateral ventricle induced progenitor cells to
re-enter the cell cycle (Hatta et al.,
2002). However, the addition of LIF did not increase the capacity
of cells from other brain regions, including the embryonic neocortex, to form
neurospheres (Gregg and Weiss,
2005) (T.Y. and Y.G., unpublished), suggesting that LIF may not be
the major regulator of STAT3 in the developing neocortex. Regardless,
endogenous ligands that induce STAT3 activation may be essential determinants
of the pool size of NPCs.
We found that STAT3 is expressed in the VZ, SVZ and the apical side of the
IMZ, and that its expression is downregulated around the time of neuronal
differentiation in the developing neocortex. The level of STAT3, as well as
that of the Tyr705-phosphorylated form of the protein, were also
high in NPC cultures in the presence of FGF2 but were reduced in association
with the induction of neuronal differentiation by removal of FGF2
(Fig. 3A). The high level of
STAT3 mRNA in the VZ, SVZ and the apical side of the IMZ could be ascribed to
the high level of STAT3 activity in this area, as it has been shown that STAT3
consists of a positive feedback loop between its activity and expression
levels (He et al., 2005;
Ichiba et al., 1998). Given
that STAT3 inhibits neuronal differentiation, the downregulation of STAT3
expression and activity probably contributes to the mechanism of neuronal
differentiation.
The interaction between the Delta-Notch and JAK-STAT pathways has
previously been reported (Chojnacki et
al., 2003; Kamakura et al.,
2004). In this study, we have shown that Dll1 is a key
target of STAT3 in both inhibition of neurogenesis and maintenance of NPCs,
which may underlie the non-cell-autonomous action of STAT3 in inhibiting
neuronal differentiation. These observations reveal a previously unrecognized
mechanism of interaction between the Delta-Notch and JAK-STAT pathways, and
they may explain, at least in part, previous findings obtained with
-secretase inhibitors or PS1, NOTCH1 or RBP-J knockout cells suggesting
that Notch signaling is required for FGF-induced maintenance of NPCs
(Chojnacki et al., 2003;
Hitoshi et al., 2002). It will
be interesting to investigate the regulation of other Notch ligands by STAT3
as well, especially because acute knockdown of DLL1 by RNAi results in bigger
effects than DLL1 gene deletion on NPC maintenance
(Grandbarbe et al., 2003)
(this study), implicating a compensatory mechanism among the Notch
ligands.
STAT genes regulate maintenance of stem cells in several systems, including
that of germline stem cells in male Drosophila as well as that of
embryonic and hematopoietic stem cells in mammals
(Kiger et al., 2001;
Tulina and Matunis, 2001). The
Notch pathway also plays an important role in inhibition of differentiation in
many systems, including the hematopoietic system
(Maillard et al., 2005). It
remains to be determined whether the regulation of Dll1 expression
and maintenance of the undifferentiated state by STAT3 demonstrated here are
also operative in stem cells of other tissues.
Supplementary materialSupplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/13/2553/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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