Stepwise differentiation of NSCs.
To delineate multiple regulatory steps in the growth and differentiation of NSCs, we used neurosphere culture of the forebrain neuroepithelium derived from E13.5 rat embryos (roughly corresponding to E11.5 in mice). When plated on the nonadhesive culture surface in the presence of FGF2 and EGF (here collectively called GFs), 4.7% ± 0.5% (n = 6) of the initially seeded cells formed clonally expanding neurospheres. The vast majority of cells comprising these neurospheres expressed nestin and Sox2, and virtually no cells (<0.1%) expressed markers for neurons or glia (Fig. ). After passage, 13.4% ± 0.6% (n = 3) of these cells were capable of forming secondary neurospheres (Fig. ; also see Fig. ). Moreover, these passaged spheres maintained the ability to give rise to neurons, astrocytes, and oligodendrocytes (Fig. ). Thus, these cells retained the capacity of self-renewal and multilineage differentiation, two cardinal properties of NSCs.
FIG. 1. Neurosphere culture of embryonic forebrain NSCs and manipulation of Notch signaling by retrovirus-mediated gene transfer. (A and A′) Bright-field (A) and fluorescence (A′) images of a neurosphere stained for nestin (red). (B) Clonal expansion (more ...)
FIG. 5. GF-independent actions of Notch in NSCs. (A and B) Regulation of self-renewal by Notch. Virus-infected cells were cultured at a clonal density (10 cells/μl) in a methylcellulose matrix for 7 days in the presence of GFs, and the number of clonal (more ...)
We next followed the time course of differentiation of these neurospheres. Upon removal of GFs, nestin- and Sox2-positive undifferentiated cells gradually decreased, and 45.8% ± 3.0% (n
= 3) of the cells became TuJ1-positive neurons at DAP2 (Fig. ). Following this early neuronal differentiation, GFAP-positive astrocytes and O4-positive oligodendrocytes emerged at later time points: 8.1% ± 0.1% and 3.4% ± 0.2% (n
= 3) of total cells became GFAP positive and O4 positive, respectively, at DAP6 (Fig. ). We observed a similar temporal sequence of neurogenesis and gliogenesis by staining for HuC/D, S100β, and galactocerebroside (data not shown). We further used three markers to detect intermediate progenitors. The proneural helix-loop-helix transcription factors Ngn2 and Mash1 and the homeodomain factor Prox1 are expressed in intermediate progenitors that emerge during differentiation of NSCs into neurons and glia (5
). Consistent with their temporal expression patterns in vivo, Ngn2-positive and Mash1-positive cells emerged at DAP1, which was followed by the peak of Prox1-positive cells at DAP2 in neurosphere culture (Fig. ). Thus, two sequential steps were distinguished along the course of differentiation of NSCs: the early transition from NSCs to intermediate progenitors and the subsequent differentiation of neurons and glia (Fig. ).
Notch regulates differentiation of NSCs.
For genetic manipulations of NSCs, we used the GFP-expressing recombinant retrovirus vector pMXIG (24
). When infected with pMXIG viruses, growing NSCs and their neuronal and glial progeny stably expressed GFP (Fig. ). Importantly, virus-infected, GFP-positive cells and noninfected, GFP-negative cells within the same culture exhibited an essentially identical capacity for self-renewal and differentiation. The overall growth and differentiation of cells were also indistinguishable between virus-infected and uninfected cultures (data not shown). Thus, the virus infection procedure did not select specific subpopulations of cells or alter the properties of NSCs.
Previous studies reported that alterations of Notch signaling did not affect the cell fate choice between neurons and glia (10
). We sought to reevaluate this issue by a combination of gain- and loss-of-function experiments. To stimulate Notch signaling, we overexpressed two constitutive-active forms of Notch1: one contained the intracellular domain of Notch1 except for the C-terminal 159 amino acid residues (ca-Notch1), whereas the other had an additional deletion of the RAM domain (ca-Notch1ΔRAM) (46
) (Fig. ). To inhibit Notch signaling, we used a dominant-negative form of the Notch receptor ligand Dll1 (dn-Dll1), in which most of the intracellular domain was deleted (47
) (Fig. ). We also used the synthetic γ-secretase inhibitor DAPT to block the ligand-dependent cleavage and activation of Notch receptors (12
Unlike control virus-infected cells, the vast majority of cells expressing ca-Notch1 remained as nestin-positive and Sox2-positive cells even after removal of GFs (Fig. ). They neither generated Mash1-positive and Prox1-positive intermediate progenitors at DAP2 (Fig. ) nor differentiated into neurons or glia at DAP6 (Fig. ). In contrast, when ca-Notch1ΔRAM was overexpressed, the percentage of GFAP-positive astrocytes among GFP-positive cells became much higher than that in the control culture at the expense of TuJ1-positive neurons and O4-positive oligodendrocytes. Inhibition of Notch signaling caused the opposite effects: cells expressing dn-Dll1 or treated with DAPT generated more Mash1-positive and Prox1-positive cells at DAP2 (Fig. ) and differentiated into neurons at a higher percentage at the expense of astrocytes (Fig. ). DAPT treatment always exerted effects similar to those of dn-Dll1 in neurosphere culture (see below), supporting the idea that it acted primarily as an inhibitor for Notch signaling in our culture. The rate of cell death, measured as the frequency of TUNEL-positive cells among total cells (2 to 3% and 18 to 20% at DAP1 and DAP6, respectively), did not significantly differ among cultures infected with different viruses, indicating that selective elimination of particular cell lineages did not account for the observed differences. Moreover, differentiation of GFP-negative, uninfected cells within the same culture occurred similarly to that in the control, demonstrating that virus-mediated modulations of Notch signaling affected the cell fate in a cell-autonomous manner. Notch ligands lacking the cytoplasmic domain, similar to dn-Dll1 used here, have been shown to inhibit Notch signaling in both cell-autonomous and non-cell-autonomous manners (18
). In our culture, however, overexpression of dn-Dll1 affected the phenotypes of virus-infected cells but not surrounding uninfected cells, demonstrating that it acted predominantly in a cell-autonomous manner.
FIG. 2. Regulation of NSC differentiation by Notch signaling. (A to C) Effect of early-step modulation of Notch signaling on differentiation of NSCs. Neurospheres were infected with retroviruses according to the early-infection protocol. Treatment with DAPT (1 (more ...)
These results suggest that Notch signaling exerts different actions at two distinct steps: the first is to block early transition from undifferentiated NSCs to intermediate progenitors, and the second is to control cell fates at a later step. To test this idea, cells were infected with viruses 2 days after induction of differentiation. In this late-infection experiment, the majority of cells passed through the early differentiation step and many of them became postmitotic neurons. Thus, the major targets for virus infection were intermediate progenitors and cells that were committed to specific lineages but not yet fully differentiated. As described above, when cells were infected with ca-Notch1 viruses before induction of differentiation, few ca-Notch1 expressing cells could become neurons or glia (2.0% ± 0.3% of total GFP-positive cells; n = 6). In contrast, when the same viruses were used for late infection, a significant fraction of ca-Notch1-expressing cells differentiated (15.5% ± 1.9%; n = 3) (Fig. ), and the vast majority of such differentiated progenies (about 95%) were astrocytes (Fig. ). Moreover, late infection with ca-Notch1ΔRAM viruses markedly increased the percentage of astrocytes, in parallel with a large (2.6-fold) increase of total differentiated cells (neurons plus glia) in culture (44.5% ± 3.1%; n = 3) (Fig. ). Conversely, dn-Dll1 suppressed astrocyte differentiation and enhanced neurogenesis under the same conditions. These results suggest that Notch signals selectively stimulate astrogenesis once NSCs are released from the early differentiation block. The marked increase of GFAP-positive cells by ca-Notch1ΔRAM also suggests that this ca-Notch1 construct not only stimulates early specification of astrocytes but also promotes subsequent expansion of astrocyte progenitors and their maturation.
Distinct signaling pathways downstream of Notch1.
In the experiments described above, two ca-Notch1 constructs exhibited significantly different activities in NSCs. We thus compared their signaling activities using two transcriptional reporters (Fig. ). We have previously shown that ca-Notch1 can both activate the transcription driven by the Hes1
enhancer and inhibit the activity of Mash1 (46
). Consistent with previous reports (2
), ca-Notch1ΔRAM activated the Hes1
enhancer-luciferase construct pHes1-Luc to the same extent as ca-Notch1 (Fig. ). Activation of this reporter depends on RBP-J (also called CBF-1) (45
), suggesting that the RBP-J-dependent signaling pathway remains active downstream of ca-Notch1ΔRAM. Nevertheless, unlike ca-Notch1, ca-Notch1ΔRAM could not inhibit the Mash1-dependent activation of the E-box-containing reporter pE7βA-Luc (Fig. ). Given that Mash1 plays a crucial role in neuronal differentiation (42
), this inhibition of Mash1 underlies, at least in part, ca-Notch1-dependent inhibition of the early differentiation of NSCs. These results can be explained by the idea that ca-Notch1 exerts its action through two distinct signaling pathways and that ca-Notch1ΔRAM is defective in activating one of these pathways. Activation of this pathway requires the RAM domain but appears to occur independently of the RBP-J/CBF-1-dependent transcriptional activation of Hes1
. These data, however, do not exclude the possibility that ca-Notch1ΔRAM was a weak allele of ca-Notch1 and that its activity was sufficient for activation of pHes1-Luc but not for inhibition of Mash1.
FIG. 3. Distinct signaling activities of ca-Notch1 and ca-Notch1ΔRAM. Activation of the Hes1 promoter-containing reporter pHes1-Luc (A) and inhibition of Mash1-dependent transcriptional activation of the E-box-containing reporter pE7βA-Luc (B) (more ...) Cross talk between Notch and GF signaling.
We next examined the relationship between Notch and GF signaling. When neurosphere cells were kept exposed to GFs, they did not differentiate into either neurons or glia even after being dissociated and cultured in monolayer (Fig. ). Like ca-Notch1, GFs suppressed differentiation at or before the step of induction of Mash1-positive intermediate progenitors (Fig. ). Unlike the results of a previous report (13
), this GF-dependent differentiation inhibition occurred under conditions in which Notch signals were blocked by either dn-Dll1 (Fig. ) or DAPT (Fig. ). Conversely, ca-Notch1 could inhibit differentiation of neurospheres in the absence of GFs (Fig. ). Thus, GFs and Notch signals appeared to be capable of blocking differentiation of NSCs independently of each other.
FIG. 4. Independent actions of Notch and GF signals on differentiation of NSCs. (A) Effect of GFs on the differentiation of NSCs. Control virus-infected cells were cultured in the presence or absence of GFs, and the percentages of marker-positive cells among (more ...)
We next examined their relationship in terms of the regulation of the self-renewal activity of NSCs. Virus-infected cells were seeded at a clonal density (10 cells/μl) into a methylcellulose matrix to prevent spontaneous cell aggregation. Subsequently, the frequencies of cells capable of forming clonal secondary neurospheres were compared among cells infected with different viruses. dn-Dll1 strongly attenuated the formation of neurospheres in the presence of GFs (39% of the control level) (Fig. ). Conversely, ca-Notch1, but not ca-Notch1ΔRAM, significantly increased the frequency of neurosphere formation in the presence of GFs (Fig. ).
GF-independent action of Notch signaling in neurosphere formation.
The above results demonstrate that both GFs and Notch signaling are required for the active self-renewing growth of NSCs. In fact, few cells formed neurospheres without GFs in control culture (0.022% ± 0.006% of initially seeded cells) (Fig. ). We found, however, that a small but significant fraction of cells expressing ca-Notch1 formed clonal colonies even in the absence of GFs (0.47% ± 0.1%; n
= 3) (Fig. ). These colonies grew slowly and were composed of nestin-positive and Sox2-positive undifferentiated cells (Fig. and data not shown). After passage, these neurospheres could form secondary spheres at a frequency similar to that for cells expanded in the presence of GFs (8.9% ± 0.6% of total cells; n
= 6). In contrast, ca-Notch1ΔRAM did not have the ability to support such GF-independent growth (Fig. ). Overexpression of the inhibitory helix-loop-helix factors Hes1, Hes5, Id1, and Id3, which are known downstream effectors for Notch signals (30
), did not fully recapitulate the activity of ca-Notch1 either (Fig. and data not shown), suggesting that molecules other than these factors are involved downstream of ca-Notch1.
The above-described neurosphere formation assays were performed at a clonal density (10 cells/μl). Under this condition, individual single cells need to survive and divide into two cells to receive Notch signals through cell-cell interactions. However, cells divide only slowly or do not survive well without GFs. Thus, it is likely that not all cells capable of forming clonal colonies can actually grow as neurospheres in such a condition. This could be a reason why more cells formed neurospheres when ca-Notch1 was expressed. To test this idea, we next asked if cells could form growing colonies at a higher frequency when they were maintained at a higher density and thereby had a higher chance to receive Notch signals during the initial period of their survival and growth. In fact, when cells were seeded at a five-times-higher density (50 cells/μl), growing colonies were formed at a much higher frequency (an 80-fold increase; 177.9 ± 14.2 colonies/1 × 104 cells) in the absence of GFs (Fig. ). Although these colonies were not necessarily clonal, they remained as undifferentiated cells and gave rise to neurons and glia when induced to differentiate (data not shown). Importantly, overexpression of dn-Dll1 and treatment with DAPT strongly suppressed the formation of these colonies, and conversely, ca-Notch1-expressing cells formed colonies at a higher frequency under this condition. These results demonstrate that the survival and growth of NSCs without GFs strongly depend on cell-cell interactions mediated by Notch signals.
A possible explanation for this GF-independent growth is that Notch regulates the expression and/or secretion of endogenous growth-promoting factors, which in turn act on NSCs in a paracrine and/or autocrine fashion. We first tested this idea by using inhibitors for FGF and EGF signaling. The tyrosine kinase inhibitors SU5402 and PD168393 selectively blocked the FGF2- and EGF-dependent formation of neurospheres, respectively (Fig. ). Neither single nor combined treatment with these inhibitors, however, inhibited the clonal expansion of ca-Notch1-expressing neurospheres in the absence of GFs (Fig. ). Thus, it is unlikely that signaling for endogenous FGF2, EGF, and/or related growth factors accounts for the Notch-dependent formation of neurospheres. Yet, it could be possible that growth-promoting factors other than the members of the FGF and EGF families are involved in Notch-dependent growth. However, in culture of ca-Notch1 virus-infected cells, stimulation of clonal growth was observed only for virus-infected cells and not for surrounding uninfected cells, suggesting that the contribution of the non-cell-autonomous action of Notch is minimum.
We found two additional GF-independent actions of Notch. To clonally expand as neurospheres, NSCs need to be maintained on the nonadhesive culture surface. When plated on the adhesive surface, cells remained undifferentiated and continued to divide, but they could not form tightly packed floating clusters even in the presence of GFs (Fig. ). Although some cells initially formed small aggregates, they eventually collapsed and dispersed on the culture surface (Fig. ). Therefore, the growth of undifferentiated cells and formation of neurosphere colonies were separable under this condition, and GFs were sufficient for the former but not for the latter. Importantly, cells expressing ca-Notch1 maintained tight cell clusters even under this condition (Fig. ). These cells formed floating colonies at a much higher frequency than control cells and kept growing as floating clusters for over 3 weeks (Fig. ). Thus, one of the crucial functions of Notch signaling independent of GFs is to enhance the cell-cell interactions of self-renewing NSCs.
FIG. 6. Control of cell adhesion and cell cycle progression by Notch signaling. (A to C) Notch-dependent clustering of clonally growing cells on the adhesive surface. The cells were cultured on the adhesive culture surface at a clonal density in a methylcellulose (more ...)
We also found that the growth of neurospheres became significantly slower when ca-Notch1 was overexpressed. In the presence of GFs, more than 50% of control cells formed clonal neurospheres with diameters larger than 50 μm at DAP7 (average diameter, 62.8 μm; n = 647) (Fig. ). In contrast, the majority of ca-Notch1-expressing cells formed much smaller spheres under the same conditions (average diameter, 40.7 μm; n = 249). Thus, strong Notch signals appear to partially counteract the growth-promoting action of GFs. In fact, when control and ca-Notch1-expressing cells were labeled with BrdU for 2 h, the percentage of BrdU-labeled cells was significantly decreased by ca-Notch1 in both the presence and absence of GFs (Fig. ). Nevertheless, the percentage of cells positive for the cyclin-dependent kinase inhibitor p27, which represented the fraction of cells that exited the cell cycle, was also lower in culture of ca-Notch1-expressing cells than in the control (Fig. ). Thus, cells with a high Notch activity divided slowly, yet a large fraction of them remained in the mitotic cycle. Altogether, these results demonstrate that Notch signaling controls multiple aspects of the self-renewal mode of growth of NSCs independently of GFs.
Cross talk between Notch and CNTF signaling in astrocyte differentiation.
As described above, Notch signaling promotes astrogenesis at the late differentiation step. The IL-6 and BMP families of cytokines also play important roles in differentiation of astrocytes (3
). We thus asked how the Notch pathway cross talks with these cytokine signals. We chose CNTF and BMP4 as representatives of IL-6 and BMP family cytokines, respectively.
As shown in previous studies, both cytokines markedly increased the percentage of GFAP-positive astrocytes among total cells (Fig. ). BMP4 also decreased the fractions of both neurons and oligodendrocytes, whereas CNTF reduced only neurons (Fig. ). Importantly, CNTF, but not BMP4, counteracted the suppression of astrogenesis by ca-Notch1 (Fig. ). In ca-Notch1-expressing cells, this CNTF-dependent induction of astrocytes did not accompany the release of the inhibition of neuronal and oligodendroglial differentiation (Fig. ). Thus, when CNTF and Notch signals were combined, NSCs differentiated selectively into astrocytes. Conversely, dn-Dll1 (Fig. ) and DAPT (Fig. ) significantly attenuated the CNTF-dependent induction of astrocytes, whereas the inhibition of neurogenesis by CNTF remained unchanged (Fig. ). Thus, CNTF-dependent induction of astrocytes appears to require endogenous Notch signals. dn-Dll1 and DAPT also inhibited astrocyte differentiation in the control culture (Fig. ). It has recently been shown that the CNTF-related factor CT-1 is secreted from neurons and acts as the major astrocyte-inducing signal (3
). Thus, the above results can be explained by the idea that endogenous CT-1 induces differentiation of astrocyte in control culture and that it requires cooperation with endogenous Notch signaling to fully exert its action.
FIG. 7. Cross talk between Notch, GF, and CNTF signaling pathways in astrocyte differentiation. (A to C) Modulation of CNTF- and BMP-dependent induction of astrocytes by Notch signaling. The percentages of GFAP-positive (A), TuJ1-positive (B), and O4-positive (more ...)
A similar cross talk was observed between CNTF and GFs. Without CNTF, GFs strongly blocked astrocyte differentiation at both low (2 ng/ml each for FGF2 and EGF) and high (20 ng/ml) concentrations (Fig. ). When combined with CNTF, however, GFs significantly augmented the CNTF-dependent induction of astrocytes. We also found that CNTF markedly decreased the frequency of neurosphere formation in the presence of GFs. Such an inhibitory effect of CNTF was observed even in cells expressing ca-Notch1 (Fig. ). Thus, CNTF blocked the Notch- and GF-dependent self-renewal of NSCs. These results suggest that the mode of cross talk between CNTF, Notch, and GF signals is distinct at the early and late differentiation steps. It appears that CNTF counteracts the differentiation block by Notch and GF signals at the early step. At the late step, however, CNTF collaborates with Notch and GFs to selectively induce astrocytes.
Regulation of STAT3 phosphorylation by Notch, GFs, and CNTF.
Recent studies have shown that the transcription factor STAT3 plays important roles in both self-renewal and astrogenesis (1
). We thus examined whether the cross talk between the Notch, GF, and CNTF pathways occurs at the level of STAT3. We examined the phosphorylation of STAT3 at the tyrosine at position 705 (Y705) and the serine at position 727 (S727), both of which are critical for STAT3-dependent transcriptional activation (20
We found that CNTF, GFs, and ca-Notch1 all induced nuclear accumulation of STAT3-pS727 (Fig. ). However, ca-Notch1ΔRAM, which lacked the ability to support GF-independent growth of NSCs, did not induce STAT3-pS727-positive cells. In contrast, STAT3 phosphorylation on Y705 was not induced by ca-Notch1 or GF treatment alone (<1.0% of total cells) and was induced only weakly by CNTF alone (15.5% ± 4.2%; n
= 3) (Fig. ). The combination of CNTF with ca-Notch1 or ca-Notch1ΔRAM, however, resulted in a marked increase of STAT3-pY705-positive cells (Fig. ). Combinatorial actions of CNTF and ca-Notch1 were also observed regarding the phosphorylation on S727 (Fig. ). These results demonstrate that the phosphorylation status of STAT3 is differentially regulated in self-renewing cells and in cells induced to differentiate into astrocytes. When Notch and GF signals maintained undifferentiated NSCs, STAT3 was phosphorylated preferentially on S727. In contrast, when CNTF signals acted on such cells in addition to Notch and GFs, STAT3 was phosphorylated on both S727 and Y705 in parallel to astrocyte differentiation. Interestingly, ca-Notch1ΔRAM, which exhibited a strong astrocyte-inducing activity, was a very weak inducer of phosphorylation of S727 and Y705 by itself but strongly augmented the CNTF-dependent response. This result reinforces the idea that Notch-dependent induction of astrocytes occurs in collaboration with endogenous CNTF-related factors such as CT-1 (3
FIG. 8. Synergistic actions of Notch, GFs, and CNTF on phosphorylation of STAT3. (A to H) Nuclear accumulation of phosphorylated STAT3 in response to Notch and CNTF signals. Cells infected with control and ca-Notch1 viruses were treated with or without CNTF for (more ...)
Lastly, we asked if similar stage-dependent regulation of STAT3 phosphorylation occurs in vivo. In the developing forebrain, NSCs at E13.5 actively proliferate and generate predominantly neurons. Astrogenesis induced by CNTF and related factors such as CT-1 begins at around E15.5, and many GFAP-positive cells differentiate at E18.5 (reference 3
and references therein). In line with this stage-dependent gliogenesis in vivo, the phosphorylation level of STAT3-Y705 was low at E13.5 but progressively increased between E13.5 and E18.5 (Fig. ). In contrast, phosphorylation of STAT3 on S727 was detected at a significant level at E13.5 and remained relatively unchanged between E13.5 and E18.5, in accordance with the persistence of proliferating NSCs throughout development.