 |
||||
|
||||
|
||||
|
||||
The Journal of Neuroscience, July 9, 2003, 23(14):6132-6140
Previous Article | Next Article
Sequential Signaling through Notch1 and erbB Receptors Mediates Radial Glia Differentiation
Brooke A. Patten,1 Jean Michel Peyrin,1 Gerry Weinmaster,2 andGabriel Corfas1 1Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, and 2Department of Biological Chemistry, University of California Los Angeles School of Medicine, Los Angeles, California 90095-1737
Abstract
Top
Abstract
Introduction
Radial glia cells both generate neurons and physically guide nascent neurons to their target destination in the cortex, and as such they are essential for CNS development. It has been proposed that in the developing cerebellum, neuronal contact induces radial glia formation, however, the mechanisms involved in this process are not well understood. Here we demonstrate that neuronal induction of radial glia formation is the result of sequential signaling through Notch1 and erbB receptors. First, Notch1 activation by neuronal contact induces the glial expression of the brain lipid binding protein (BLBP) and erbB2 genes. Interestingly, two different signaling pathways mediate these effects of Notch1 on transcription, BLBP expression being dependent on Su(H), whereas erbB2 is regulated by a yet unidentified Notch1 pathway. The subsequent increase in erbB2 receptor expression makes the glia more responsive to neuronal NRG, which then induces the morphological transformation into radial glia. Thus, these results unveil some of the mechanisms underlying radial glia formation.
Key words: Notch1; Su(H); erbB; cerebellum; glia; BLBP
Introduction
Cell migration is a major step in the development of the vertebrate CNS. Newborn neurons, which are generated in the germinal layers of the neural tube, must move to their final destination in the cortex and other areas of the CNS, where they establish appropriate synaptic connections. These cell movements are critical for normal brain development and function. Radial glia constitute a population of specialized cells that play critical roles in this neuronal migration. Radial glia are present from early stages of neural tube formation, and they possess thin processes that, in most cases, span the entire thickness of the developing cerebral wall. Early studies indicate that radial glia fibers act as scaffolds to guide migrating newborn neurons from their birthplaces to their final destinations in the developing cortex (Rakic and Sidman, 1970; Hatten and Mason, 1990
Studies on cerebellar development indicate that contact between neurons and glia is a critical event for the formation of radial glia. Using cocultures of cerebellar granule cells and astroglia, it has been shown that these neurons induce astrocytes to undergo a morphological, functional, and molecular differentiation into radial glia. For example, granule cell contact induces cerebellar astrocytes to extend a process along which neurons can migrate (Hatten, 1985
Using cerebellar cells, we previously demonstrated that neuron–glia contact induces the morphological differentiation of radial glia via neuregulin (NRG)–erbB receptor signaling, and that erbB receptor signaling in radial glia is necessary for neuronal migration (Rio et al., 1997
The Notch receptors and their ligands, molecules best known for influencing cell fate decisions through direct cell–cell contact (Nye and Kopan, 1995
The most widely accepted model for Notch signaling proposes that after ligand binding to Notch, the receptor undergoes proteolytic cleavages within its extracellular and intramembrane sequences, releasing the Notch intracellular domain (NICD) from the membrane. A group of proteins collectively known as the CSL proteins, which include Suppressor of Hairless [Su(H)] in Drosophila and its homologous genes in Xenopus [X-Su(H)], mammals (RBP-J/CBF1/KBF2) and Caenorhabditis elegans (LAG-1) are important mediators of Notch signaling. These proteins bind DNA and act as transcriptional repressors, but through direct interaction with the cytoplasmic domain of Notch they are converted into transcriptional activators. Accordingly, Notch is a clear example of a transmembrane receptor that functions not only in ligand binding but also as a signal transducer that directly activates gene transcription.
To investigate the roles of Notch1 signaling in the induction of radial glia formation, we used the well characterized in vitro system of cerebellar astrocytes and granule neuron cocultures. Here we report that contact with cerebellar granule neurons activates Notch1 signaling in cerebellar astroglia and that activation of Notch1 is sufficient to induce the morphological differentiation of radial glia and the expression of the radial glia gene BLBP. Analysis of the interactions between Notch1 and erbB receptor signaling showed that Notch1 signaling is upstream of erbB signaling and that Notch1-induced radial glia formation is mediated by its effects on erbB receptor expression and function. Our results show that radial glia differentiation requires sequential activation of Notch1 and erbB receptor signaling and that the Notch1 effects on radial glia differentiation require signaling through two intracellular pathways, one that requires CSL and one that does not.
Materials and Methods
Histology and immunostaining. Long–Evans postnatal day 6 (P6) rat pups (Charles River Laboratories, Wilmington, MA) were fixed by intracardial perfusion with 4% paraformaldehyde in PBS, and the cerebellum was dissected, cryoprotected, and cut in 20 µm parasagittal sections. Tissue sections were blocked with 3% BSA and 0.1% Triton X-100 in PBS for 1 hr at room temperature, followed by incubation with mouse anti-glial fibrillary acidic protein (anti-GFAP; Boehringer Mannheim, Indianapolis, IN) and an affinity-purified rabbit anti-Notch1 polyclonal antibody (Ab 93–4, 1:100) (Shawber et al., 1996bCells in culture were fixed in 4% paraformaldehyde for 10 min, rinsed with PBS, blocked as above, and then labeled with a rabbit anti-GFAP (Dako, Carpinteria, CA) and/or mouse monoclonal anti-erbB4 antibody (NeoMarkers, Fremont, CA). Nuclei were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) or SYBR Green (Molecular Probes).
Cell culture. Primary astroglial and neuronal cells were purified from Long–Evans P6 rat cerebella (Charles River) by selective preplating. Cerebella were excised, and the meninges were removed in Dulbecco's PBS (Invitrogen, Gaithersburg, MD), under a dissecting microscope. The tissue was cut into small pieces and incubated in 1% trypsin (Sigma, St. Louis, MO) and 0.02% deoxyribonuclease (DNase; Worthington, Free-hold, NJ) in PBS for 10 min at 37°C. After centrifugation, the pellet was triturated using successively decreasing bore size fire-polished glass Pasteur pipettes in astrocyte media (DMEM with high glucose), 10% fetal bovine serum (FBS), and 2 mM L-glutamine (Invitrogen) with 0.02% DNase. Cell were pelleted, resuspended in fresh medium, plated onto two 100 mm tissue culture dishes, and incubated at 37°C for 20 min to allow fibroblasts to attach to the plate. Then, the unattached cells (neurons and glia) were removed from the dishes and plated onto one poly-D-lysine (PDL; 0.5 µg/ml; BD Biosciences) -coated dish. After 1 hr incubation at 37°C, unattached cells (primarily neurons) were removed, and the dish was washed gently with PBS to remove any loosely attached cells. The remaining attached cells, primarily glia, were incubated in astrocyte media. The supernatant from the first PDL-coated dish was plated onto a second PDL-coated dish, and the steps described above were repeated to generate another dish of glial cells. The washout of the second PDL dish was used as the source of purified granule cells. The neurons were centrifuged and resuspended in astrocyte media with 1% N2 supplement (Invitrogen). Glia were passaged 3–4 d later, a step that removed any remaining neurons, producing a glial culture >95% pure. All cells were incubated at 37°C with 5% CO2. Parental fibroblasts (SR
) and Jagged1-expressing (SN3T) (Lindsell et al., 1995
Plasmids. FCDN1 and OCDN1 cDNAs were cloned into mammalian expression vector pEF1a-BOS (Mizushima and Nagata, 1990
Recombinant NRG. Recombinant epidermal growth factor (EGF)-like domain of rat NDF
1 (NDF
Transfections. Primary astrocytes at 70% confluence were transfected with Fugene 6 reagent (Boehringer Mannheim), according to the recommended protocol. To detect the transfected cells, cells were cotransfected with a plasmid encoding green fluorescent protein (GFP) at a molar ratio of 3:1 (plasmid:GFP plasmid). The level of coexpression of the genes of interest and GFP was assessed by immunostaining with antibodies against erbB4 (NeoMarkers) or Notch1 (Ab 93–4, 1:100) (Shawber et al., 1996b
Luciferase assay. Luciferase assays were performed 2 d after transfection using the Dual Assay Luciferase kit (Promega). Cotransfected TK–renilla luciferase was used to normalize samples for transfection efficiency and sample handling. Cells were lysed, and Luciferase activity was measured following the recommended protocol.
Astroglial morphology assay. Glia were plated at 10,000 cells/200 mm 2, and freshly dissociated neurons were added at a ratio of 10:1. The radial glia morphology was determined as described in Rio et al. (1997
For glia–fibroblast coculture experiments, P6 cerebellar astroglia were plated at 30,000 cells/200 mm 2 in astrocyte media. Twenty-four hours later, control (SR
Quantitative Western blot analysis. Primary astrocytes grown to 70% confluency were transfected with FCDN1 or the control plasmid (PBOS). Two days later cells were lysed in radioimmunoprecipitation assay buffer, and samples (6 µg of protein of each) were separated by SDS-PAGE on 4–20% gels (Invitrogen), transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), and probed with a rabbit polyclonal anti-erbB2 C terminus antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-284) or a rabbit polyclonal anti-BLBP antibody (a gift of N. Heintz) followed by an HRP-conjugated anti-rabbit antibody (Jackson ImmunoResearch). Blots were developed using Western Lightning chemiluminescence reagent (PerkinElmer Life Sciences, Emeryville, CA), and images were captured with the Fujifilm (Fuji, Tokyo, Japan) Intelligent Dark Box II LAS-1000 plus. Quantitation of band intensities was performed using IP Lab Gel H software. After the erbB2 and BLBP images were obtained, the membranes were reprobed with anti-actin (Oncogene Sciences, Uniondale, NY) or anti-GAPDH antibodies (Chemicon, Temecula, CA). The intensity of the actin or GAPDH bands was used for normalization of the erbB2 and BLBP signals. Both normalizers gave identical results.
Statistical analysis. Statistical significance for the astroglial morphological assays was determined by
2 using the Statview program. Statistical significance for the glia–fibroblast morphological assays was determined by Student's t test. Statistical significance for the Luciferase assays was determined by Wilcoxon Signed Rank test. Statistical significance for the quantitative Westerns was determined by paired t test.
Results
Notch1 and Jagged1 expression in the developing cerebellum
To determine the possible roles of Notch1 in postnatal cerebellar development and the migration of cerebellar granule cells along Bergmann radial glia, we studied the pattern of expression of Notch1 and its ligand, Jagged1. At P6, a time of maximal granule cell migration, Notch1 immunoreactivity was concentrated in Bergmann glia fibers, colocalizing with immunostaining for GFAP (Fig. 1). This is in agreement with in situ hybridization studies showing that Notch1 mRNA appears to be localized to Bergmann glia (Irvin et al., 2001
View larger version (50K):
[in this window]
[in a new window]
Figure 1. Notch1 is expressed by Bergmann glia fibers in the developing cerebellum. Parasagittal sections through the P6 rat cerebellum stained with antibodies against GFAP (green) and Notch1 (red). The overlay image demonstrates colocalization of GFAP and Notch1 staining in Bergmann radial glia. Scale bar, 250 µm.
Granule neurons activate Notch signaling in cerebellar glia
To determine if neuronal contact activates Notch1 signaling in cerebellar astrocytes, we measured the effects of neuron–glia contact on the Notch-dependent transcriptional activation of the CSL reporter construct. We also tested if these effects are blocked by a dominant-negative Suppressor of Hairless [DN-Su(H)], a mutant Su(H) that interacts with Notch but cannot bind DNA (Wettstein et al.,1997
View larger version (18K):
[in this window]
[in a new window]
Figure 2. Contact with granule cells activates Notch signaling in cerebellar glia. A, Purified P6 cerebellar glia were cotransfected with plasmids encoding the CBF1-Luciferase reporter and with either DN-Su(H) or a control plasmid. Twenty-four hours later, purified P6 cerebellar granule cells (5 x 104 or 5 x 105) were added to the culture. The next day cells were lysed, and Luciferase activity was measured. The results represent the mean ± SEM of three independent experiments. Asterisks mark which conditions are significantly different from the control. *p < 0.05; **p < 0.005; ***p < 0.0005. AU, Arbitrary units. B, Purified P6 cerebellar glia were cotransfected with plasmids encoding the CBF1-Luciferase reporter and FCDN1, DN-Su(H), or a control plasmid, or both FCDN1 and DN-Su(H) together. Two days later, cells were lysed, and Luciferase activity was measured. The results represent the mean ± SD of two independent experiments. Asterisks mark which conditions are significantly different from the control. *p < 0.003; **p < 0.0005. C, Purified P6 cerebellar glia were transfected with a plasmid encoding the CBF1-Luciferase reporter. Twenty-four hours later, purified P6 cerebellar granule cells or NRG (1 nM) were added to the culture. The next day cells were lysed, and Luciferase activity was measured. The results represent the mean ± SD of two independent experiments. Asterisks mark which conditions are significantly different from the control, p < 0.005.
To study further the specificity of the CBF1 reporter activation, we tested if NRG-erbB receptor signaling, a signaling pathway shown to mediate granule cell–glia interactions (Rio et al., 1997
Notch signaling induces radial glia formation
It has been well documented that contact with granule cells induces cerebellar astroglia in culture to adopt radial glia morphology (Hatten, 1985
View larger version (55K):
[in this window]
[in a new window]
Figure 3. Activated Notch1 and Jagged1 induce radial formation. A, Purified P6 cerebellar glia were transfected with a plasmid encoding either FCDN1 or a control plasmid. Twenty-four hours later, purified P6 cerebellar granule cells (1 x 105) were added, and the next day glial morphology was examined. Radial glia were identified as GFAP(+) cells with at least one thin process longer than 50 µm. The results represent the mean ± SEM of four independent experiments. Asterisks mark which conditions are significantly different from the control. *p < 0.005; **p < 0.0001. B, Images show representative glia under control conditions, with neurons (white arrows indicate neuronal nuclei), with NRG (1 nM), or transfected with a plasmid encoding FCDN1. Glia were stained with antibodies against GFAP (green), and nuclei were stained with Hoechst (blue). The morphology of the radial glia is similar under all stimuli. Scale bar, 20 µm. C, Jagged1-expressing or parental fibroblasts were added to cultures of purified P6 cerebellar glia. Twenty-four hours later, radial glia morphology was examined. The results represent the mean ± SEM of three independent experiments; p = 0.008. D, Images show representative glia cocultured with parental fibroblasts or Jagged1-expressing fibroblasts (white arrows indicate fibroblast nuclei). Scale bar, 20 µm. Glia were stained with antibodies against GFAP (green), and nuclei were stained with Hoechst (blue).
Because Northern blot analysis indicated that cerebellar granule cells express Jagged1, we tested if this ligand by itself could induce radial glia formation. Cerebellar astroglia were cocultured either with Jagged1-expressing or parental fibroblasts, and their morphology was quantified. Contact with Jagged1-expressing cells induced morphological changes consistent with radial glia, suggesting that Jagged1-induced Notch signaling is sufficient to induce radial glia formation (Fig. 3C,D). There were no significant differences between the levels of radial glia morphology resulting from expression of FCDN1 and contact with Jagged1-expressing cells (p = 0.466).
Both Notch and erbB receptor signaling are necessary for neuronal-induced radial glia formation
To determine if Notch1 signaling is necessary for the neuronal induction of radial glia morphology, we used two published Notch1 signaling antagonists; dominant-negative forms of Notch1 (OCDN1) and Suppressor of Hairless (DN-Su(H)) (Wettstein et al., 1997
View larger version (14K):
[in this window]
[in a new window]
Figure 4. Notch and erbB receptor signaling are both necessary for neuronal induction of radial glia. A, Purified P6 cerebellar glia were transfected with plasmids encoding DN-Su(H), DN-erbB4, OCDN1, or a control plasmid. Twenty-four hours later, purified P6 granule neurons were added, and morphology was examined the next day. The results represent the mean ± SEM of four independent experiments. Asterisks mark which conditions are significantly different from the neuronal treatment. *p < 0.004; **p < 0.0007. B, Purified P6 cerebellar glia were transfected with plasmids encoding DN-Su(H), FCDN1, or a control plasmid. Two days later morphology was examined. The results represent the mean ± SEM of three independent experiments. Asterisks mark which conditions are significantly different from the control. *p < 0.004. C, Purified P6 cerebellar glia were transfected with plasmids encoding either FCDN1 or a control plasmid. Twenty-four hours later, purified P6 granule neurons or NRG (1 nM) was added, and morphology was examined the next day. The results represent the mean ± SEM of three independent experiments. Asterisks mark which conditions are significantly different from the neuronal treatment. *p < 0.02; **p < 0.0005; ***p < 0.0001.
View larger version (17K):
[in this window]
[in a new window]
Figure 5. Notch1 and erbB act sequentially to induce radial glia formation. A, Purified P6 cerebellar glia were transfected with either a plasmid encoding OCDN1 or a control plasmid. Twenty-four hours later, NRG (1 nM) was added to the culture, and morphology was examined the next day. The results represent the mean ± SEM of three independent experiments. Asterisks mark which conditions are significantly different from the control; p < 0.0003. B, Purified P6 cerebellar glia were transfected with plasmids encoding DN-erbB4, or FCDN1, or both DN-erbB4 and FCDN1 together, or a control plasmid. Twenty-four hours later, glial morphology was examined. The results represent the mean ± SEM of three independent experiments. The asterisk marks which condition is significantly different from the control; p < 0.008.
Independent activation of erbB or Notch1 signaling is capable of inducing radial glia formation, however to a lesser extent than that elicited by neuronal contact. Because neurons express ligands for both of these receptors, we tested if simultaneous activation of both pathways is similar to the effects of neurons. Cerebellar astroglia were transfected with a control plasmid and 24 hr later treated with neurons or NRG or transfected with the plasmid encoding FCDN1 and either left untreated or treated with NRG. While independent activation of the each of these pathways, Notch1 or erbB, induced radial glia formation to a lesser extent than that induced by neuronal contact, simultaneous activation of both pathways resulted in a robust response that was indistinguishable from the effects of neurons (Fig. 4C). In similar manner, stimulation with soluble NRG and coculture with Jagged1-expressing cells mimicked the effects of neurons (data not shown).
Notch and erbB receptors act sequentially to induce radial glia formation
The results presented above indicated that both Notch1 and erbB receptor signaling pathways are necessary for the induction of radial glia morphology by neuronal contact. Moreover, coactivation of both pathways simultaneously led to a greater level of radial glia formation than independent activation of each pathway. A likely explanation for these observations is that Notch and erbB function in the same cascade of events that occur subsequent to neuron–glia contact. For example, one pathway could be necessary for the effective activation of the second one, to direct radial glia formation. To test this idea and to define the order in which these signaling molecules are activated to direct glial differentiation, we determined if blockade of one pathway prevents induction of radial glia through the other. When cerebellar glia were transfected with the plasmid encoding OCDN1, the ability of NRG to induce radial glia formation was unaffected (Fig. 5A). In contrast, coexpression of DN-erbB4 blocked the ability of FCDN1 (Fig. 5B) and Jagged1 (data not shown) to induce radial glia formation. These results are consistent with the idea that the Notch1 signaling pathway is upstream of erbB receptor signaling during neuronal induction of radial glia formation.Neurons activate erbB2 expression in cerebellar glia through Notch1 signaling
The epistasis observed between Notch1 and erbB signaling suggested that Notch1 signaling might regulate some aspects of erbB signaling. For example, it is possible that Notch1 signaling induces erbB receptor expression in the glia, which in turn makes the astroglia more responsive to NRG. To test this possibility, we determined whether granule cells induce the expression of erbB2 in the glia, and if the induction is dependent on Notch1 signaling. A plasmid encoding the 4.5 kb erbB2 upstream sequences (White and Hung, 1992
View larger version (20K):
[in this window]
[in a new window]
Figure 6. Notch1 signaling mediates the neuronal induction of erbB2 expression in glia. A, Purified P6 cerebellar glia were cotransfected with plasmids encoding the erbB2-Luciferase reporter and either OCDN1 or a control plasmid. Twenty-four hours later, purified P6 granule neurons were added to the culture, and the next day cells were lysed and Luciferase activity measured. The results represent the mean ± SEM of five independent experiments. The asterisk marks which condition is significantly different from the control; p < 0.028. B, Purified P6 cerebellar glia were cotransfected with plasmids encoding the erbB2-Luciferase reporter and FCDN1, DN-Su(H), or a control plasmid. Twenty-four hours later, NRG (1 nM) or forskolin (5 µm) was added to the culture. The next day cells were lysed, and Luciferase activity was measured. The results represent the mean ± SEM of at least three independent experiments. Asterisks mark which conditions are significantly different from the control; p < 0.04. C, Purified P6 cerebellar glia were transfected with either FCDN1 or a control plasmid, lysed 48 hr later, and equal amounts of protein were subjected to quantitative Western blot analysis with rabbit polyclonal anti-erbB2 and mouse monoclonal anti-actin antibodies for normalization. D, Intensities of the erbB2 immunoreactivity normalized to the actin control. The data represent the mean ± SEM of four independent experiments; p < 0.007.
To further characterize the effects of Notch1 activation on erbB2 expression, we tested if this signaling pathway leads to increases of endogenous glial erbB2 protein. Glia were transfected with FCDN1 or a control plasmid and incubated for 2 d. Then, cells were lysed and erbB2 protein levels examined by quantitative Western blot analysis. FCDN1 expression induced a 1.75 ± 0.23-fold (p < 0.007) increase of erbB2 protein in glia (Fig. 6C,D). Because the transfection efficiency for glia in these experiments was 30%, this most likely reflects a 3.5-fold increase of erbB2 protein in the transfected cells. In these cell extracts we also tested for the possibility that expression of FCDN1 leads to increases in erbB activation, using phospho-erbB2 and phospho-tyrosine antibodies. These experiments did not detect Notch1-induced changes in erbB2 phosphorylation (data not shown). However, because ligand induced-erbB receptor phosphorylation is transient, lasting <1 hr, it was expected that chronic Notch1 signaling would not result in significant long-term erbB2 phosphorylation.
Neuronal-induction of BLBP expression in glia is mediated by Notch activation
Previous studies have shown that neuronal contact upregulates the expression of BLBP, a gene expressed by radial glia that has been proposed to be important for neuronal migration (Feng et al., 1994
View larger version (20K):
[in this window]
[in a new window]
Figure 7. Notch1 signaling mediates the neuronal induction of BLBP promoter transcription in glia. A, Purified P6 cerebellar glia were cotransfected with plasmids encoding the BLBP-Luciferase reporter and FCDN1, OCDN1, DN-Su(H), or a control plasmid. Twenty-four hours later, purified P6 granule neurons or Jagged1-expressing fibroblasts were added to the culture, and the next day cells were lysed and Luciferase activity was measured. The results represent the mean ± SEM of at least three independent experiments. Asterisks mark which conditions are significantly different from the control. *p < 0.028; **p < 0.018. B, Purified P6 cerebellar glia were cotransfected with plasmids encoding the BLBP-Luciferase reporter and FCDN1, DN-erbB4, or a control plasmid. Twenty-four hours later, purified P6 granule neurons or NRG (1 nM) was added to the culture. The next day cells were lysed, and Luciferase activity was measured. The results represent the mean ± SEM of three independent experiments. Asterisks mark which conditions are significantly different from the control. *p<0.04. C, Purified P6 cerebellar glia were transfected with either FCDN1 or a control plasmid, lysed 48 hr later, and equal amounts of protein were subjected to quantitative Western blot analysis with rabbit polyclonal anti-BLBP and mouse monoclonal anti-actin antibodies for normalization. D, Intensities of the BLBP immunoreactivity normalized to the actin control. The data represent the mean ± SEM of three independent experiments; p = 0.046.
Studies by Anton et al. (1997
To further characterize the effects of Notch1 activation on BLBP expression we tested if this signaling pathway leads to increases in endogenous glial BLBP protein. Glia were transfected with FCDN1 or a control plasmid and incubated for 2 d. Then, cells were lysed, and BLBP protein levels were examined by quantitative Western blot analysis. FCDN1 expression induced a 1.66 ± 0.18-fold (p = 0.046) increase of BLBP protein in glia (Fig. 7C,D). Because the transfection efficiency for glia in these experiments was 30%, this most likely reflects a 3.1-fold increase of BLBP protein in the transfected cells.
Discussion
Radial glia play critical roles in brain development, acting as neuronal precursors (Malatesta et al., 2000Our results show that cerebellar granule cells induce astrocytes to become radial glia by sequentially activating the Notch and the erbB receptor signaling pathways. Together, these signaling events promote the molecular, morphological, and functional differentiation of these cells (Fig. 8). Our data suggest a role for Notch1 signaling in radial glia formation that involves the transcriptional activation of radial glia-specific genes as well as erbB receptors. In contrast, the role of erbB receptor signaling appears to be the induction of morphological transformation. However, the possibility that erbB receptor signaling has effects on the expression of radial glia proteins, either by transcriptional or post-transcriptional mechanisms, remains open. Although our conclusions are based on results obtained with cerebellar cells, other observations (Anton et al., 1997
View larger version (19K):
[in this window]
[in a new window]
Figure 8. A model for neuronal induction of radial glia by sequential signaling through Notch and erbB pathways. Initial contact by a Jagged1-expressing neuron activates astrocytic Notch receptors. Notch signaling then induces expression of BLBP and erbB2 in the glia. The increase in erbB receptor expression makes the astrocyte more responsive to neuron-derived NRG, which subsequently induces the glia to adopt a radial morphology and to support neuronal migration.
DN-erbB4 has also been demonstrated to act very specifically, blocking signaling through erbB2, erbB3, and erbB4 but not through the EGF receptor (erbB1) (Prevot et al., 2003
The finding that Notch1 activation is sufficient to induce radial glia formation, but that this requires erbB receptor signaling, suggests that Notch1 signaling, in some manner, regulates or induces erbB receptor signaling. This could be the result of a Notch1-induced increase in astrocyte expression of either erbB receptors, erbB receptor ligands, or both. Our results suggest that a Notch1-mediated induction of erbB receptor expression is part of this process. Whereas we only analyzed the effects of Notch1 signaling on erbB2 expression, it is possible that Notch1 signaling is involved in the regulation of erbB3 or erbB4 expression as well. Like erbB2, these erbB receptors are expressed by cerebellar and cortical radial glia (Anton et al., 1997
These previously unknown interactions between Notch1 and erbB receptor signaling may also be significant for other cell types in which both Notch1 and erbB receptors have important biological effects, including muscle, oligodendrocytes, and Schwann cells. Notch and erbB signaling influence in similar manner the differentiation of neural crest cells into Schwann cells (Shah et al., 1994
Several studies showed that Notch and erbB receptors as well as their ligands are expressed in the postnatal cerebellum. For example, Bergmann glia express mRNA for all Notch receptors (Irvin et al., 2001
Until now, the only radial glial gene whose expression had been shown to be induced by neuronal contact was BLBP (Feng and Heintz, 1995
BLBP is not only a useful marker for radial glia but it is also an important protein for granule cell migration, given the observation that anti-BLBP antibodies block the migration of granule cells along radial glia fibers (Feng et al., 1994
A significant difference between Bergmann glia and cortical radial glia is that although the latter disappear after migration is completed, Bergmann glia persist in the adult cerebellum, maintaining their radial morphology. However, Bergmann glia cease to express BLBP after migration is finished. Irvin et al. (2001
It is clear that during the development of the vertebrate nervous system several extracellular signaling molecules contribute to particular cellular events, such as glial or neuronal differentiation. However, in most cases, the hierarchies of these signals, or the order in which they need to act, are often difficult to establish. Our results show that sequential signaling through Notch1 and then erbB receptors mediates the induction of radial glia formation by neuronal contact. Similar mechanisms may underlie the functions of Notch1 and erbB receptors in other developmental events, and sequential signaling of other extracellular molecules may serve a variety of processes such as axonal and dendritic outgrowth and pathfinding or synapse formation.
Footnotes
Received Mar. 19, 2003; revised May. 13, 2003; accepted May. 16, 2003.This work was supported in part by National Institute of Neurological Disorders and Stroke Grant R01 NS35884 (G.C.), The EJLB Foundation (G.C.), National Alliance for Research on Schizophrenia and Depression (G.C.), Mental Retardation Research Center Grant NIH P30-HD 18655 (G.C.), ARSEP (J.M.P.), and predoctoral National Institutes of Health training Grant T-32 AG00222 (B.P.). We thank Sally Temple for valuable discussions, Nathaniel Heintz for the BLBP promoter and antibody, and Phillip Leder for the erbB2 promoter.
*B.A.P. and J.M.P. contributed equally to this work.
Correspondence should be addressed to Dr. Gabriel Corfas, Division of Neuroscience, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail: gabriel.corfas{at}tch.harvard.edu.
J. M. Peyrin's present address: Laboratoire d'Immunologie des Tumeurs, Faculté de Pharmacie (Université Paris XI), 5 rue Jean Baptiste Clément, 92296 Chatenay Malabry Cedex, France.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236132-09$15.00/0
References
Alvarez-Buylla A, Theelen M, Nottebohm F (1990) Proliferation "hot spots" in adult avian ventricular zone reveal radial cell division. Neuron 5: 101–109.[ISI][Medline]
Anton ES, Marchionni MA, Lee KF, Rakic P (1997) Role of GGF/neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex. Development 124: 3501–3510. s[Abstract]
Canoll PD, Musacchio JM, Hardy R, Reynolds R, Marchionni MA, Salzer JL (1996) GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron 17: 229–243.[ISI][Medline]
Chanas-Sacre G, Rogister B, Moonen G, Leprince P (2000) Radial glia phenotype: origin, regulation, and transdifferentiation. J Neurosci Res 61: 357–363.[ISI][Medline]
Culican SM, Baumrind NL, Yamamoto M, Pearlman AL (1990) Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes. J Neurosci 10: 684–692.[Abstract]
Endo Y, Osumi N, Wakamatsu Y (2002) Biomodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development. Development 129: 863–873.
Feng L, Heintz N (1995) Differentiating neurons activate transcription of the brain lipid-binding protein gene in radial glia through a novel regulatory element. Development 121: 1719–1730.[Abstract]
Feng L, Hatten ME, Heintz N (1994) Brain lipid-binding protein (BLBP): a novel signaling system in the developing mammalian CNS. Neuron 12: 895–908.[ISI][Medline]
Gaiano N, Nye JS, Fishell G (2000) Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26: 395–404.[ISI][Medline]
Hatten ME (1985) Neuronal regulation of astroglial morphology and proliferation in vitro. J Cell Biol 100: 384–396.
[Abstract/Free Full Text] Hatten ME (1999) Central nervous system neuronal migration. Annu Rev Neurosci 22: 511–539.[ISI][Medline]
Hatten ME, Mason CA (1990) Mechanisms of glial-guided neuronal migration in vitro and in vivo. Experientia 46: 907–916.[ISI][Medline]
Hsieh JJ, Henkel T, Salmon P, Robey E, Peterson MG, Hayward SD (1996) Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol Cell Biol 16: 952–959.[Abstract]
Hunter KE, Hatten ME (1995) Radial glial cell transformation to astrocytes is bidirectional: regulation by a diffusible factor in embryonic forebrain. Proc Natl Acad Sci USA 92: 2061–2065.
[Abstract/Free Full Text] Irvin DK, Zurcher SD, Nguyen T, Weinmaster G, Kornblum HI (2001) Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development. J Comp Neurol 436: 167–181.[ISI][Medline]
Leavitt BR, Hernit-Grant CS, Macklis JD (1999) Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons. Exp Neurol 157: 43–57.[ISI][Medline]
Lindsell CE, Shawber CJ, Boulter J, Weinmaster G (1995) Jagged: a mammalian ligand that activates Notch1. Cell 80: 909–917.[ISI][Medline]
Malatesta P, Hartfuss E, Gotz M (2000) Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development 127: 5253–5263.[Abstract]
Mizushima S, Nagata S (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18: 5322.
[Free Full Text] Morrison SJ, Perez SE, Qiao Z, Verdi JM, Hicks C, Weinmaster G, Anderson DJ (2000) Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101: 499–510.[ISI][Medline]
Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409: 714–720.[Medline]
Nofziger D, Miyamoto A, Lyons KM, Weinmaster G (1999) Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts. Development 126: 1689–1702.[Abstract]
Nye JS, Kopan R (1995) Developmental signaling. Vertebrate ligands for Notch. Curr Biol 5: 966–969.[ISI][Medline]
Prevot V, Rio C, Cho GJ, Ma YJ, Neville C, Rosenthal N, Heger S, Ojeda SR, Corfas G (2003) Normal female sexual development requires NRG-erbB receptor signaling in hypothalamic astrocytes. J Neurosci 23: 230–239.
[Abstract/Free Full Text] Rakic P, Sidman RL (1970) Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans. J Comp Neurol 139: 473–500.[ISI][Medline]
Redmond L, Oh SR, Hicks C, Weinmaster G, Ghosh A (2000) Nuclear Notch1 signaling and the regulation of dendritic development. Nat Neurosci 3: 30–40.[ISI][Medline]
Rieff HI, Raetzman LT, Sapp DW, Yeh HH, Siegel RE, Corfas G (1999) Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells. J Neurosci 19: 10757–10766.
[Abstract/Free Full Text] Rio C, Rieff HI, Qi P, Khurana TS, Corfas G (1997) Neuregulin and erbB receptors play a critical role in neuronal migration. Neuron 19: 39–50.[ISI][Medline]
Shah NM, Marchionni MA, Isaacs I, Stroobant P, Anderson DJ (1994) Glial growth factor restricts mammalian neural crest stem cells to a glial fate. Cell 77: 349–360.[ISI][Medline]
Shawber C, Boulter J, Lindsell CE, Weinmaster G (1996a) Jagged2: a serrate-like gene expressed during rat embryogenesis. Dev Biol 180: 370–376.[ISI][Medline]
Shawber C, Nofziger D, Hsieh JJ, Lindsell C, Bogler O, Hayward D, Weinmaster G (1996b) Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122: 3765–3773.[Abstract]
Solecki DJ, Liu X, Tomoda T, Fang Y, Hatten ME (2001) Activated Notch2 signaling inhibits differentiation of cerebellar granule neuron precursors by maintaining proliferation. Neuron 31: 557–568.[ISI][Medline]
Tanaka M, Kadokawa Y, Hamada Y, Marunouchi T (1999) Notch2 expression negatively correlates with glia differentiation in the postnatal mouse brain. J Neurobiol 41: 524–539.[Medline]
Wang S, Sdrulla AD, diSibioG, Bush G, Nofziger D, Hicks C, Weinmaster G, Barres BA (1998) Notch receptor activation inhibits oligodendrocyte differentiation. Neuron 21: 63–75.[ISI][Medline]
Weinmaster G (1997) The ins and outs of notch signaling. Mol Cell Neurosci 9: 91–102.[ISI][Medline]
Wen D, Suggs SV, Karunagaran D, Liu N, Cupples RL, Luo Y, Janssen AM, Ben-Baruch N, Trollinger DB, Jacobsen VL (1994) Structural and functional aspects of the multiplicity of Neu differentiation factors. Mol Cell Biol 14: 1909–1919.
[Abstract/Free Full Text] Wettstein DA, Turner DL, Kintner C (1997) The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124: 693–702.[Abstract]
White MR, Hung MC (1992) Cloning and characterization of the mouse Neu promoter. Oncogene 7: 677–683.[Medline]
Yamamoto N, Yamamoto S, Inagaki F, Kawaichi M, Fukamizu A, Kishi N, Matsuno K, Nakamura K, Weinmaster G, Okano H, Nakafuku M (2001) Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor. J Biol Chem 276: 45031–45040.
[Abstract/Free Full Text]
This article has been cited by other articles:
A. D. Nelson, M. Suzuki, and C. N. Svendsen
A High Concentration of Epidermal Growth Factor Increases the Growth and Survival of Neurogenic Radial Glial Cells Within Human Neurosphere Cultures
Stem Cells, February 1, 2008; 26(2): 348 - 355.
[Abstract] [Full Text] [PDF]
R. L. Dillon, S. T. Brown, C. Ling, T. Shioda, and W. J. Muller
An EGR2/CITED1 Transcription Factor Complex and the 14-3-3{sigma} Tumor Suppressor Are Involved in Regulating ErbB2 Expression in a Transgenic-Mouse Model of Human Breast Cancer
Mol. Cell. Biol., December 15, 2007; 27(24): 8648 - 8657.
[Abstract] [Full Text] [PDF]
H.T. Ghashghaei, J. M. Weimer, R. S. Schmid, Y. Yokota, K. D. McCarthy, B. Popko, and E.S. Anton
Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex
Genes & Dev., December 15, 2007; 21(24): 3258 - 3271.
[Abstract] [Full Text] [PDF]
R. H. Friedel, G. Kerjan, H. Rayburn, U. Schuller, C. Sotelo, M. Tessier-Lavigne, and A. Chedotal
Plexin-B2 Controls the Development of Cerebellar Granule Cells
J. Neurosci., April 4, 2007; 27(14): 3921 - 3932.
[Abstract] [Full Text] [PDF]
