The presence of FGF2 signaling determines whether β-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells

Developmental Biology 268 (2004) 220 – 231 www.elsevier.com/locate/ydbio

The presence of FGF2 signaling determines whether h-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells Nipan Israsena, Min Hu, Weimin Fu, Lixin Kan, and John A. Kessler * Department of Neurology, Northwestern University’s Feinberg School of Medicine, Chicago, IL 60611-3008, USA Received for publication 17 October 2003, revised 22 December 2003, accepted 23 December 2003

Abstract Neural stem cells proliferate and maintain multipotency when cultured in the presence of FGF2, but subsequent lineage commitment by the cells is nevertheless influenced by the exposure to FGF2. Here we show that FGF2 effects on neural stem cells are mediated, in part, by h-catenin. Conversely, the effects of h-catenin in neural stem cells depend in part upon whether there is concurrent fibroblast growth factor (FGF) signaling. FGF2 increases h-catenin signaling through several different mechanisms including increased expression of h-catenin mRNA, increased nuclear translocation of h-catenin, increased phosphorylation of GSK-3h, and tyrosine phosphorylation of h-catenin. Overexpression of h-catenin in the presence of FGF2 helps to maintain neural progenitor cells in a proliferative state. However, overexpression of h-catenin in the absence of FGF2 enhances neuronal differentiation. Further, chromatin immunoprecipitation (ChIP) assays demonstrate that both h-catenin and Lef1 bind directly to the neurogenin promoter, and luciferase reporter assays demonstrate that h-catenin is directly involved in the regulation of neurogenin 1 and possibly other proneural genes when neural stem cells are cultured in the presence of FGF2. We suggest that the balance between the mitogenic effects and the proneural effects of h-catenin is determined by the presence of FGF signaling. D 2004 Elsevier Inc. All rights reserved. Keywords: h-catenin; FGF2 signaling; Neuronal differentiation

Introduction Neural stem cell survival, proliferation, lineage commitment, and differentiation are all regulated by fibroblast growth factor (FGF) signaling. FGF2 and FGFR1 are both expressed within the ventricular epithelium during early stages of neurogenesis (Ghosh and Greenberg, 1995; Temple and Qian, 1995; Vaccarino et al., 1999a), and FGF2 knockout mice display a severe depletion of progenitor cells in the dorsal ventricular epithelium with a reduction in cortical neuron number (Raballo et al., 2000). FGF2 treatment of cultured neural progenitor cells provides mitogenic and trophic support and also influences progenitor cell fate choice (Maric et al., 2003; Palmer et al., 1999; Qian et al., 1997). FGFs also act in concert with other growth factors to regulate neural stem cells. For example, members of the Wnt family of secreted glycoproteins are usually found in * Corresponding author. Department of Neurology, Northwestern University’s Feinberg School of Medicine, Ward Building 10-185, 303 East Chicago Avenue, Chicago, IL 60611-3008, USA. Fax: +1-312-503-0872. E-mail address: [email protected] (J.A. Kessler). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.12.024

the proximity of high FGF synthesis domains during neural development and act in cooperation with FGFs in controlling cell fate (Gunhaga et al., 2003; Kuschel et al., 2003; Viti et al., 2003). There are extensive data indicating that Wnt or h-catenin signaling enhances neurogenesis in the developing embryo (Baker et al., 1999; Dorsky et al., 1998; Fujita et al., 2001; Hyodo-Miura et al., 2002; Megason and McMahon, 2002; McGrew et al., 1999; Molenaar et al., 1998; Yasumoto et al., 2002). Further, h-catenin, an important mediator of canonical Wnt-signaling pathways, plays an important role in regulating proliferation or differentiation of neural stem cells (Chenn and Walsh, 2002, 2003; Viti et al., 2003; Zechner et al., 2003). Overexpression of constitutively active h-catenin in neural stem cells in vivo increases neurogenesis primarily by decreasing cell cycle exit of neural progenitors (Chenn and Walsh, 2002). In Wnt signaling, binding of Wnt proteins to their specific cell-surface receptors, the frizzleds, activates the cytoplasmic protein disheveled (Dsh), which in turn leads to a reduction in activity of glycogen synthase kinase (GSK-3) (Cook et al., 1996). GSK-3h constitutively phosphorylates h-catenin and targets it to a degradation pathway, and GSK-

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3h also phosphorylates and activates Axin, which binds to and promotes h-catenin degradation (Jho et al., 2002, Willert et al., 1999). Thus, activation of Wnt receptors in the canonical pathway leads to accumulation of h-catenin in the cytoplasm because of reduced degradation. h-catenin then forms complexes with the transcription factor Lef/Tcf and enters the nucleus to regulate target genes. In this complex, Lef/Tcf provides the DNA binding domain, while h-catenin contributes the transactivation domain (reviewed by Willert and Nusse, 1998). The FGF and Wnt-signaling pathways may cross regulate each other by multiple mechanisms. For example, FGF1 can phosphorylate and deactivate GSK-3h, leading to accumulation of the signaling pool of h-catenin (Hashimoto et al., 2002) and can also affect the distribution of h-catenin through modulation of cadherin (Huber and Weis, 2001). FGF2 induces Lef/Tcf-dependent transcription in human endothelial cells (Holnthoner et al., 2002). Further, both pathways influence expression of each other’s signaling components in a cell-type specific manner (Ciruna and Rossant, 2001; El-Hariry et al., 2001; Imai et al., 2002; Kawakami et al., 2001; Kratochwil et al., 2002). In this study, we investigate how FGF2 affects components of the Wnt-signaling pathway in neural progenitor cells and examine effects of h-catenin in regulating neural progenitor proliferation and differentiation. We find that FGF2 signaling increases the pool of h-catenin in cultured progenitor cells through several different mechanisms, and that hcatenin signaling plays an important role in maintaining neural potential by directly and indirectly controlling expression of proneural genes.

Materials and methods Retrovirus production and infection of cultures Virus was produced by double transfection of 293FT cells (Invitrogen) with pCLE-IRES-GFP and pVSVG constructs. Viral supernatant was collected for 3 days and 100fold concentrated by ultracentrifugation at 25,000  g for 1 h and 30 min. For viral infection, concentrated virus was added to the medium after neural progenitors were dissociated and replated in fresh medium containing 10 ng/ml FGF2 for 24 h. Forty-eight hours after infection, the neurospheres were dissociated and 104 cells were plated on PDLor laminin-coated coverslips.

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carefully dissected and dissociated by incubation with 0.05% EDTA or trypsin (Life Technologies) for 5 min followed by mechanical dissociation. After a wash, the cells were spun down and resuspended in DMEM/F12 (Life Technologies) supplemented with N2 (Life Technologies), B27 (Life Technologies), 2 Ag/ml heparin, 100 U/ ml penicillin, 100 Ag/ml streptomycin, and 2 mM Lglutamine (Life Technologies). FGF2 (Becton Dickenson) was then added to the medium at the final concentration of 10 ng/ml. Cells were grown in 1  10 cm Petri dishes for 5 days before passage at 37jC and 5% CO2. Neurosphere numbers and sizes were determined by analyzing photomicrographs of 10 representative areas of each set of cultures. P19 embryonal carcinoma cells (ATCC) were maintained in MEM alpha medium (Life Technologies) supplemented with 7.5% newborn calf serum (Life Technologies), 2.5% fetal bovine serum (Life Technologies), 100 U/ml penicillin, 100 Ag/ml streptomycin, and 2 mM L-glutamine (Life Technologies) in humidified 5% CO2. For experiments involving retinoic acid induction, cells were trypsinized and then plated at 1  105 cells/ml in MEM alpha supplemented with 5% FBS in 10 cm Petri dishes. All trans retinoic acid (RA) was then added to the medium (1mM) and cells were allowed to aggregate. After 2 days with or without retinoic acid treatment, nuclear and cytoplasmic proteins were collected. Immunocytochemistry Cells were fixed with 4% paraformaldehyde for standard indirect immunofluorescence staining. Briefly, fixed cover slips were rinsed in PBS, permeabilized, then blocked with 2% normal goat serum or donkey serum for 30 min before incubation with primary antibody overnight at 4jC. Primary antibodies used were as follows: mouse anti-nestin (1:400; Pharmingen); chicken anti-GFP (1:2000; Chemicon), antih-III tubulin (1:400; Sigma), and anti-GFAP (1:400; Sigma). The following secondary antibodies were used to visualize cells: Alexa 488 and 568 conjugated goat antimouse IgG (1:200; Molecular Probes) and FITC-conjugated donkey anti-chicken IgG (Southern Biotech). Coverslips were incubated in PBS containing Hoechst 33258 solution for 10 min for nuclear staining, then washed and mounted with Prolong antifade reagent (Molecular Probes). In studies of proliferation of the P19 cell line, the cells were pulsed with BrdU (Sigma) for 4 h and then immunostained with anti-BrdU antibody (Chemicon).

Cell culture For neural progenitor cells, timed pregnant CD1 mice were sacrificed by cervical dislocation and embryos were removed. FGF-generated neurospheres were then established and differentiated as previously described (Zhu et al., 1999). Briefly, the lateral and median ganglionic eminence regions of embryonic day 13.5 mice were

Co-immunoprecipitation and Western blotting Nuclear and cytoplasmic extracts from treated and untreated neural progenitors and P19 cells were collected using NE-PER reagent (Pierce) according to the manufacturer’s instructions. For immunoprecipitation analyses, the cells

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were lysed in a lysis buffer (50 mM Tris –HCL, 150 mM NaCl, 1% Nonidet P40, and 0.5% sodium deoxycholate) supplemented with ‘‘complete’’ protease inhibitors (Roche) for 10 min with agitation at 4jC. Insoluble material was removed by centrifugation at 14,000  g for 10 min, and the supernatants were used for Western blot analysis or immunoprecipitation. Protein concentration was determined with the BCA protein assay kit (Pierce). Equal amounts of total proteins were used for immunoprecipitations. For immunoprecipitations, cell lysates were incubated with anti-h-catenin (Santa Cruz) or normal mouse IgG with protein G-agarose overnight at 4jC. The bound proteins were washed five times to reduce the nonspecific signal and then eluted in the same amount of elution buffer. Bound proteins and cell lysates were resolved by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% milk in PBST then incubated with anti-phosphotyrosine antibody (1:200; Upstate Biotechnology), anti-h-catenin antibody (1:400; Santa Cruz), antiphosphorylated GSK (1:1000; Cell signaling), or anti-total GSK (1:1000; Cell signaling) overnight at 4jC. Membranes were then washed three times and incubated with the appropriate HRP-conjugated secondary antibody for 1 h. After three washes with PBST, the signals were developed with ECL (Perkin). Luciferase dual reporter assays The ratio between activities of fly luciferase driven by different regulatory sequences of proneural genes and the constitutive expression of Ranilla driven by the herpes simplex virus thymidine kinase promoter (pRL-TK) was measured using the dual luciferase reporter system (Promega). Chromatin immunoprecipitation (ChIP) assay Co-immunoprecipitation experiments examining proteinDNA interactions were performed using the chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology). For P19 cells, cells were seeded at the density of 1  105 cells/ml. Cells were allowed to form aggregates for 2 days in the presence or absence of retinoic acid before cross linking with formaldehyde as described below. For neural progenitors, after cells were split and cultured for 4 days as neurospheres, cells were washed two times with PBS and then cultured in FGF-free medium for 4 h before 50 ng/ml of FGF2 were added in the experimental group. After 1 h of FGF treatment, cells were fixed by adding formaldehyde directly to culture medium to a final concentration of 1% and incubating for 10 min at 37jC. The medium was removed and the cells were washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 Ag/ml aprotinin, and 1 Ag/ml pepstatin A). The cells were scraped into a conical tube, pelleted for 4 min at

2000 rpm at 4jC, resuspended in 200 Al of SDS lysis buffer containing the protease inhibitors, and incubated for 10 min on ice. The lysate was sonicated to shear DNA to lengths between 200 and 1000 bp and then diluted 10-fold in ChIP dilution buffer (with protease inhibitors). Two milliliters of the diluted cell pellet suspension was precleared with 80 Al of salmon sperm DNA or protein A agarose—50% slurry for 1 h at 4jC with agitation to reduce nonspecific background. The agarose was pelleted by brief centrifugation and the supernatant fraction was collected. Anti-h-catenin antibody (Santa Cruz) or normal mouse IgG was added to 2 ml of the supernatant fraction and was incubated overnight at 4jC with rotation. Agarose was then pelleted by gentle centrifugation (700 –1000 rpm at 4jC, approximately 1 min), and the supernatant containing unbound nonspecific DNA was carefully removed. The protein A agarose –antibody –protein – DNA complexes were washed with high salt, low salt, and LiCl solution for 10 min each on a rotating platform. The complex was then eluted from the agarose by adding 250 Al elution buffer to the pelleted protein A complex, vortexing briefly, and incubating the mixture at room temperature for 15 min with rotation. The agarose was pelleted and the supernatant fraction was carefully transferred to another tube. Ten microliters of 5 M NaCl was added to the combined eluates and the protein-DNA cross-links were reversed by heating at 65jC for 4 h. The eluate was then used as the template for PCR, and the PCR products were detected on a regular agarose gel. DNA binding assays Nuclear extracts were prepared from neural progenitor cells or P19 cells using NE-PER (Pierce). Protein levels in the extract were measured by BCA methods and equivalent amounts were diluted in binding buffer (10 mM Tris, 50 mM KCL, 1 mM DTT, pH 7.5, 5% glycerol), and 0.3 Ag of 5V biotinylated target DNA was added. Following a 20-min incubation, AMACS streptavidin-conjugated microbeads (Miltenyl Biotec) were added, and samples were incubated for an additional 15 min. Samples were then put through magnetic columns (Miltenyl Biotec) and washed four times with binding buffer. Bound protein was then eluted with elution buffer and run on an SDS-PAGE gel as described above. Antibodies used in the Western blot were anti-Lef1 antibody (Santa Cruz Biotechnology) and anti-SOX1 (from Professor Hisato Kondoh, Japan).

Results FGF2 increases b-catenin mRNA expression and translocation of b-catenin to the nucleus in neural progenitor cells To study how FGF2 signaling affects neural progenitor cells, we first used a DNA microarray technique to compare

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gene expression profiles between E14 mouse neural progenitor cells cultured in low concentrations (10 ng/ml) versus high concentrations of FGF2 (50 ng/ml). We found that h-catenin mRNA was elevated eightfold at 2 h and 14fold at 6 h in the cells treated with higher doses of FGF2. The up-regulation of h-catenin mRNA by increased doses of FGF2 was confirmed by Northern blot analysis (Fig. 1A). Up-regulation of h-catenin mRNA might not be indicative of an up-regulation of h-catenin signaling for several reasons. The fraction of h-catenin that can enter the nucleus and regulate target genes is very small compared to the amount of total h-catenin protein inside the cell, and the ratio between the amount of h-catenin in the signaling pool and the amount that is in the cell adhesion complex is also under the influence of multiple factors. Therefore, to directly assess the signaling pool of h-catenin, we investigated whether FGF2 treatment increases h-catenin translocation to the nucleus. After neural progenitors were grown as neurospheres for 4 days in regular medium containing 10 ng/ml FGF2, cells were washed with PBS and cultured in medium without FGF2 for 6 h before 0, or 20 ng/ml of FGF2were added to the medium. Nuclear extracts were harvested at 30 min and 1 h after FGF2 was reintroduced. We found that FGF2 treatment significantly increased the level of nuclear h-catenin within 30 min of treatment (Fig. 1B).

translation of h-catenin. We therefore examined the effects of FGF2 on tyrosine phosphorylation of h-catenin, which has been shown to reduce h-catenin affinity to the adherin complex (Balsamo et al., 1996). We found that FGF2 treatment increased the level of tyrosine-phosphorylated h-catenin while the level of total h-catenin protein was not significantly altered (Fig. 1C). Unlike serine or threonine phosphorylation, which would target h-catenin for degradation, increased tyrosine phosphorylation would be expected to make a greater percentage of total cellular hcatenin available for the nuclear signaling pool (Balsamo et al., 1996; Piedra et al., 2001) as was noted (Fig. 1B). The activity of GSK-3h, which constitutively targets hcatenin for the degradation pathway, can be inhibited by growth factor-mediated phosphorylation (see Doble and Woodgett, 2003). Therefore, we also examined whether FGF2 affects GSK-3h phosphorylation in neural progenitor cells. The level of phosphorylated GSK-3h increased within an hour of FGF2 treatment while the level of total GSK-3h did not change (Fig. 1D). This also would be expected to increase the nuclear signaling pool of h-catenin as was observed (Fig. 1B).

FGF signaling induces phosphorylation of GSK-3b and tyrosine phosphorylation of b-catenin

We next examined the role of h-catenin and FGF2 signaling on neural progenitor cell proliferation and lineage commitment by using retroviral vectors to overexpress hcatenin in E14 neural progenitor cells cultured in the presence or absence of FGF2. When h-catenin overexpressing progenitors were kept in medium that contained FGF2

Since FGF2 treatment promoted nuclear translocation of h-catenin so rapidly, it seemed likely that the effects of FGF2 did not solely reflect increased transcription and

Effects of b-catenin on neural progenitor cells cultured in the presence or absence of FGF2

Fig. 1. FGF2 increases the signaling pool of h-catenin mRNA through multiple mechanisms. (A) FGF2 promotes expression of h-catenin mRNA. Northern blot analysis of E14 neurosphere-derived neural progenitor cells cultured for 2 h in the presence of either 10 or 50 ng/ml of FGF2. Note that increasing the dose of FGF2 increased levels of h-catenin mRNA. (B) FGF2 signaling increases the nuclear pool of h-catenin. E14 neural progenitor cells were cultured in the absence of FGF2 for 6 h. The cells were then treated with FGF2 (20 ng/ml) or buffer for 30 or 60 min, and Western blot analyses were performed on nuclear lysates with anti-h-catenin antibody. BCA assays ensured that equal amounts of protein were loaded onto each lane. (C) FGF2 increases the proportion of h-catenin that is tyrosine phosphorylated. E14 neural progenitor cells were cultured in the absence of FGF2 for 6 h and FGF2 was then added (0 or 50 ng/ml). Cell lysates collected 1 h later were immunoprecipitated with anti-h-catenin antibody and were then blotted with either anti-h-catenin antibody (left) or anti-phosphotyrosine antibody (right). (D) FGF2 increases the proportion of GSK-3h that is phosphorylated. E14 neural progenitor cells were cultured in the absence of FGF2 for 6 h. FGF2 was then added (0, 10, or 50 ng/ml), protein was collected 1 h later, and protein lysates were blotted with anti-phospho-GSK-3h (top) or anti-GSK-3h (bottom).

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and passaged, the cells generated significantly more secondary spheres (Fig. 2A). It is known that virtually all spheres are clonally derived when neural progenitor cells are plated at a density < 5  104 cells/ml (Hulspas et al., 1997; Tropepe et al., 1999), indicating that a greater proportion of the h-catenin overexpressing cells was able to reenter cell cycle and self-renew than control cells. However, the size of neurospheres generated from h-catenin-overexpressing progenitor cells and control viral-infected progenitor cells did not differ significantly, and BrdU incorporation by the neurospheres over an 8-h period also did not differ. This

Fig. 3. h-catenin promotes neuronal differentiation of P19 cells. (A) P19 cells treated with retinoic acid have higher levels of cytosolic and nuclear hcatenin. P19 cells were cultured either with or without retinoic acid (RA, 0.5 AM) for 2 days and the protein lysates were analyzed by Western blots using an anti-h-catenin antibody. BCA assays indicated that equal amounts of protein were loaded onto each pair of lanes. (B and C) h-catenin promotes neuronal differentiation of P19 cells without retinoic acid. P19 cells were transfected with either the control GFP construct or wild-type hcatenin, replated and allowed to differentiate under low serum conditions without retinoic acid. Control cells (B) displayed no detectable immunoreactivity for hIII tubulin (red), whereas 5 – 10% of P19 cells overexpressing h-catenin (C) expressed hIII-tubulin after 4 days.

Fig. 2. In the presence of FGF2, h-catenin promotes reentry into cell cycle and self-renewal of neural progenitor cells, whereas in the absence of FGF2 it promotes neuronal differentiation. (A) Neural progenitor cells that overexpress h-catenin generate more secondary neurospheres in the presence of FGF2. Retrovirus-infected progenitor cells were cultured in suspension for 2 days, and the resulting neurospheres were dissociated and plated at a density of 200 cells/200 Al in 96-wells plates. Secondary neurospheres were quantified 5 days after plating. Overexpression of hcatenin or of a constitutively active form of h-catenin (DN) significantly increased the number of neurospheres generated compared to control (empty vector) but no difference in the size of the spheres was detectable. ** Differs from control by ANOVA, P < 0.025. (B) Neural progenitor cells that overexpress h-catenin generate more neurons when FGF2 is withdrawn. E14 neural progenitors were infected with a retrovirus containing h-catenin, a constitutively active form of h-catenin (DN) or empty virus (pCLE). Infected progenitor cells were cultured in suspension for 2 days before neurospheres were dissociated and plated on PDL coated coverslips at a density of 2  104 cells/ml in the absence of FGF2. Cells were fixed 96 h after plating and immunostained for nestin (progenitor cells), hIII-tubulin (neurons), GFAP (astrocytes), and CNPase (oligodendrocytes). Cells were counted with the observer blinded to the identity of the cultures. Overexpression of either wild-type h-catenin or constitutively active h-catenin increased the percentage of neurons at the expense of nestin positive cells. Overexpression of wild-type h-catenin or constitutively active h-catenin did not alter cell survival, indicating a specific effect on lineage commitment *Differs from control (pCLE), P < 0.025.

suggests that h-catenin increased the probability of reentry of progenitor cells into cell cycle but not the rate of proliferation, similar to the findings of Chenn and Walsh, (2002). In contrast, when cells overexpressing h-catenin were plated onto a substrate in the absence of FGF2, there was a significant increase in the number of hIII tubulin neurons (Fig. 2B). Overexpression of a constitutively active form of h-catenin resulted in even more enhancement of neuronal lineage commitment. There was no significant change in the percent of cells that differentiated into GFAP immunoreactive astrocytes or CNPase immunoreactive oligodendrocytes. However, the percent of cells expressing nestin was significantly reduced by overexpression of hcatenin, suggesting that these cells had differentiated into neurons (Fig. 2B). Importantly, overexpression of h-catenin did not alter total cell numbers, indicating that under these conditions, it regulated lineage commitment but not proliferation. It should be noted that since only about 30% of the cells were transduced by the virus (based on GFP expression), the effects of h-catenin may actually be more pronounced than reported in this experiment. When only GFP-expressing cells were considered, more than 65% of the cells expressed hIII tubulin compared to the less than 19% in vector controls. By contrast, h-catenin did not alter lineage commitment when the cells were cultured in the presence of FGF2. Thus, h-catenin promotes either main-

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tenance of the proliferative state or neuronal lineage commitment depending upon the cellular context (presence of FGF2). The role of b-catenin in neural induction of P19 cells The foregoing experiments indicated that h-catenin exerts proneuronal effects on cells that have already committed to a neural lineage. To define the role of hcatenin in cells that have not yet committed to a neural lineage, we next examined the effects of h-catenin in P19 cells, a pluripotent cell line that can differentiate into cell

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types of all three germ layers with the appropriate stimuli. P19 cells can be induced to become neurons with retinoic acid treatment, and Wnt signaling has been reported to be involved in this process (Papkoff, 1994, Tang et al., 2002). We found that retinoic acid treatment increased the level of both nuclear and cytosolic h-catenin in aggregated P19 cells (Fig. 3A). Further, overexpression of h-catenin was sufficient to promote neuronal differentiation (h-tubulin III immunoreactivity) without retinoic acid treatment (Fig. 3B). However, the effects of h-catenin on lineage commitment were overcome by growing cells in high-serum containing medium. Cell cycle analysis of h-catenin trans-

Fig. 4. h-catenin transfection alters P19 cell proliferation in a serum-dependent manner. P19 cells were transfected with either the control GFP construct or wild-type h-catenin and purified by FACS sorting for GFP+ transfected cells. Two days later, the cells were analyzed for stage of cell cycle by PI-staining followed by FACS sorting. In growth medium, h-catenin slightly increased the percentage of cells in a proliferative state; whereas in serum-free medium, hcatenin overexpressing P19 cells exited the cell cycle more than the control group.

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factors in serum that are responsible for its mitogenic effects on P19 cells are not clear. Nevertheless, these results suggest that while h-catenin has mitogenic effects, it is not by itself sufficient to maintain cells in the proliferative state and requires other growth factors (e.g., serum or FGF2) for these effects to predominate. In contrast, in situations where growth factors are limited (e.g., serum-free medium, no FGF2), the proneuronal

Fig. 5. h-catenin stimulates neurogenin 1 promoter activity through Lef/ Tcf-dependent mechanisms. (A) Overexpression of h-catenin increases neurogenin 1 promoter activity in 293 cells. 293 cells were transfected with a control luciferase vector and a neurogenin 1 promoter-driven luciferase vector according to the Dual Reporter Luciferase Assay system. These cells were then transfected either with control vector, a h-catenin expression construct, or a constitutively active Notch expression construct (Nicd). Cells transfected with h-catenin showed a sixfold increase and cells transfected with Nicd showed a fivefold decrease in the level of activity of the neurogenin 1 promoter. Deletion of the hairy binding site on the neurogenin 1 promoter (D Hairy-BD) led to increased activation by hcatenin and a reduction in the level of suppression by Nicd. *Differs from control by ANOVA, P < 0.01, ** P < 0.03. (B) Deletion of two potential Lef/Tcf binding sites on the neurogenin 1 promoter abolishes the effects of h-catenin. P19 cells were transfected with 0.1 Ag of wild-type (WT) neurogenin 1 promoter or a mutated promoter in which a fragment of 12 nucleotides containing two potential Lef/Tcf binding sites had been deleted (DLef1-BD) and pRL-TK for an internal control. Activation of wild-type neurogenin 1 promoter was observed as early as 12 h after transfection, whereas the mutated promoter did not respond to h-catenin overexpression. *Differs from control by ANOVA, P < 0.025.

fected cells also demonstrated that h-catenin overexpression did not affect cell cycle progression in high-serum containing medium, but that it promoted cell cycle exit in serum-free medium (Fig. 4). In high-serum medium, 38.6% of control cells were in G 0/G 1 compared to 36.1% of h-catenin overexpressing cells. In serum-free medium, 48.2% of control cells were in G0/G1 compared to 57.6% of cells overexpressing h-catenin. Further, in high-serum medium overexpression of h-catenin, increased BrdU incorporation by 14% but conversely reduced BrdU incorporation by 18% in the serum-free medium. The

Fig. 6. h-catenin and Lef1 bind to the neurogenin 1 promoter in neural progenitor cells. (A) DNA binding assays demonstrate that Lef1 in nuclear lysates from cortical neurospheres can selectively bind to the potential Lef/ Tcf binding sites on the neurogenin 1 promoter. Nuclear extracts from E14 cortical neurospheres were incubated with a biotin-labeled 50-bp long nucleotide fragments of the neurogenin 1 promoter that contain two Lef/Tcf binding sites or a 4-bp mutation of the Lef/Tcf binding site along with streptavidin-linked magnetic beads before being passed through a magnetic field. Trapped proteins were then eluted, run on an SDS gel, and blotted with anti-Lef1 or anti-Sox1 antibody. Nuclear extracts from cortical neurospheres contain Lef1, which binds to the fragment of the wild-type neurogenin promoter but not to the mutated site. The related transcription factor SOX1 cannot bind to this fragment. (B) h-catenin is bound to the neurogenin promoter in E14 progenitor cells cultured with FGF2. E14 progenitor cells were cultured in the presence or absence of FGF2 (10 ng/ ml), and protein or nucleic acid complexes were collected and analyzed by chromatin immunoprecipitation (ChIP). Anti-h-catenin antibody was used to pull down h-catenin containing nucleoprotein complexes. Normal mouse IgG was used for the control. The DNA was then dissociated from the complex and analyzed by PCR using primers to detect the neurogenin 1 promoter. Note that h-catenin is bound to the neurogenin 1 promoter only when the neural progenitors were cultured in the presence of FGF2. (C) hcatenin is bound to the neurogenin promoter in P19 cells induced to become neurons by retinoic acid treatment. P19 cells were grown in the presence or absence of retinoic acid (RA, 0.5 AM), and nucleoprotein complexes were analyzed by ChIP as in part A. Note that h-catenin was bound to the neurogenin 1 promoter when cells were grown with RA but not in the absence of RA.

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effects of h-catenin predominate in both pluripotent (P19) and multipotent (neurosphere) cells. Regulation of neurogenin 1 Because we found that h-catenin can promote neuronal differentiation of both P19 cells and neural progenitor cells, we used a luciferase reporter construct containing 1.6 kb of the neurogenin 1 regulatory element to determine whether h-catenin is directly involved in the regulation of proneural gene expression. In fact, h-catenin expression elevated levels of neurogenin 1 transcription more than sixfold in a dual luciferase reporter assay (Fig. 5A). Sequence analysis of the neurogenin 1 promoter or enhancer (Murray et al., 2000) using Matinspector software revealed multiple potential Lef/Tcf binding sites. Deletion of two of the Lef/Tcf binding sites negated the effects of h-catenin on this reporter (Fig. 5B). We then sought to determine whether this protein –DNA interaction occurs in normal tissue. Using a DNA binding assay, we found that Lef1 present in nuclear extracts of cortical neural progenitor cells bound to the 50-bp DNA sequence from neurogenin 1 promoter, which contains two putative Lef/Tcf binding sites (Fig. 6A). Mutation of the Lef/Tcf site in the neurogenin 1 promoter abolished binding, indicating the specificity of the Lef1 binding to this site. As an additional control for the specificity of the binding, we found that Sox1 (which shares a closely related binding sequence with Lef/Tcf), in nuclear extracts from neural progenitor cells cannot bind to this sequence (Fig. 6A). Thus, Lef1 is bound to the neurogenin promoter in neural stem cells that are cultured in the presence of FGF2. We next examined whether h-catenin regulates neurogenin 1 expression by itself binding to this regulatory element. Chromatin immunoprecipitation studies indicated that when neural progenitors were cultured in medium containing FGF2, h-catenin was part of a complex bound to the neurogenin promoter (Fig. 6B). In contrast, in the absence of FGF2, we could not detect an association between h-catenin and the neurogenin 1 promoter. In P19

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cells, we also detected an association between h-catenin and the neurogenin 1 promoter when P19 cells were treated with retinoic acid but not in untreated cells (Fig. 6C). Thus, hcatenin is directly involved in regulation of neurogenin 1 expression in both neural progenitor cells and P19 cells. Effects of b-catenin on other bHLH factors We also tested the effects of h-catenin on the regulatory regions of two other proneural genes, neurogenin 2 and mash1, both of which also contain potential Lef/Tcf binding sites. We found that h-catenin strongly up-regulates the neurogenin 2 reporter construct (Fig. 7A) but that it has only a small effect on the mash1 reporter construct (Fig. 7C). h-catenin also activates the promoter or enhancer of myoD (Fig. 7B), consistent with the known muscleinducing effects of overexpression of h-catenin in P19 cells (Petropoulos and Skerjanc, 2002). We conclude that h-catenin can activate a number of fate determination genes of the bHLH family and that this effect of h-catenin is very likely to play an important role in the regulation of stem cell lineage commitment.

Discussion The present study demonstrates that FGF2 increases hcatenin signaling in neural progenitor cells through several different mechanisms, including increased expression of hcatenin mRNA, increased nuclear translocation of h-catenin, increased phosphorylation of GSK-3h, and tyrosine phosphorylation of h-catenin. h-catenin is important both for regulation of cell cycle and for maintaining neural potential. Importantly, the mitogenic effects of h-catenin predominate in conditions that foster proliferation whereas the proneural effects are evident in conditions that promote exit from cell cycle. Further, we found that h-catenin binds directly to the promoters of several proneural genes and activates their expression, which may underlie the proneural effects of h-catenin.

Fig. 7. h-catenin can activate multiple fate determination bHLH factors. Luciferase reporter assays demonstrate that h-catenin overexpression increases promoter activity of neurogenin 2 (A), myoD (B), and to the lesser degree mash1 (C). 293 cells (80% confluence) were transfected with 0.1 Ag of reporter plasmid and 0.4 Ag of either control vector or vector that contains the h-catenin gene under the CMV promoter. Firefly luciferase activity was normalized with the cotransfected Ranilla luciferase under the control of thymidine kinase promoter. Shown are the means and SD of three experiments for each promoter. *Differs from control at P < 0.001. **Differs from control at P < 0.025.

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Involvement of b-catenin in FGF signaling in neural stem cells We found that treatment with FGF2 up-regulated levels of h-catenin mRNA in a dose-dependent fashion and increased levels of nuclear h-catenin within 30 min of treatment. We conclude that FGF2 is capable of increasing the signaling pool of h-catenin and that some h-catenin target genes may be activated by FGF2 in neural stem cells. The increased levels of h-catenin after FGF2 treatment reflected, in part, increased phosphorylation of GSK3h. This observation is consistent with the prior finding that FGF1 can similarly promote phosphorylation or inactivation of GSK-3h in neural cells through the PI3K pathway (Hashimoto et al., 2002). The mechanisms of regulation of GSK-3h by FGF and Wnt are different, providing a means by which Wnt and FGF can exert synergistic effects on h-catenin activity (reviewed by Ali et al., 2001). Tyrosine phosphorylation of catenin or cadherin has been correlated with instability of the complex (Balsamo et al., 1996), thereby increasing the pool of free hcatenin that can potentially translocate to nucleus. We found that FGF2 treatment increases tyrosine phosphorylation of h-catenin (Fig. 1C) in neural progenitor cells. This would be expected to increase the transcriptional activity of the h-catenin/Tcf complex by increasing interactions of h-catenin with TATA-box binding protein (Piedra et al., 2001). Function of b-catenin in neural stem cells h-catenin mRNA is highly expressed in the VZ of normal animals (Chenn and Walsh, 2002). Our findings, combined with previous studies demonstrating that FGF2 and FGFR1 are highly expressed in the VZ (Vaccarino et al., 1999b), suggest that h-catenin signaling may play a physiologic role in the VZ by acting downstream of FGF signaling. What is the role of h-catenin signaling in neural progenitor cells? Recent studies suggest that h-catenin signaling is capable of regulating neural stem cell proliferation and the size of the cerebral cortex in vivo. Transgenic mice overexpressing a stabilized form of h-catenin in neural progenitors had an abnormally large number of neural precursor cells, as well as an increase in cortical surface area (Chenn and Walsh, 2002). This reflected decreased exit of the progenitor cells from cell cycle rather than an increase in the rate of proliferation (Chenn and Walsh, 2002). Since this experiment involved overexpression of h-catenin in an artificial way, some concern has been raised regarding the relevance of this to normal cortical development (Vogel, 2002). Nevertheless, this study and others (Zechner et al., 2003) make it clear that under some conditions, h-catenin exerts predominantly mitogenic effects, a conclusion supported by our studies. However does h-catenin simply regulate reentry into cell cycle or does it have other effects as well? We used two separate culture systems to address

this question: P19 embryocarcinoma cells and neural progenitors cultured as neurospheres from E14 mouse cortex. We found that levels of nuclear h-catenin increased markedly during retinoic acid-induced neural induction of P19 cells. Further, overexpression of h-catenin promoted neuronal differentiation without retinoic acid treatment under low serum conditions. Our results are consistent with various other reports that suggest that Wnt or catenin promotes differentiation of these cells. Further, overexpression of Wnt-1 in P19 cells resulted in the loss of expression SSEA1, a marker of undifferentiated P19 cells (Smolich and Papkoff, 1994). A recent study also demonstrated that that Wnt3a can promote neural differentiation in P19 cells, whereas overexpression of Axin, which leads to down regulation of h-catenin signaling, results in a blockage in neuronal differentiation (Lyu et al., 2003). It should be noted that we also observed an increase in neuronal differentiation in both embryonic stem cells and P19 cells treated with Wnt3a (unpublished data). Importantly, we observed an increase in neuronal differentiation only when h-catenin-transfected P19 cells were cultured in low serum conditions at low density. In high serum-containing medium, the transfected cells remained epithelial-like and h-catenin staining showed membrane localization. Cell cycle analysis also confirmed that in conditions that promote cell cycle exit, h-catenin enhanced exit from cell cycle and differentiation instead of promoting proliferation. Our results suggest that while hcatenin signaling is known to increase levels of cell cycle progression proteins such as cyclin D1, it must work in concert with other epigenetic stimuli to maintain cells in cell cycle. By contrast, situations or molecules that promote cell cycle exit (e.g., low density and lack of serum) will enhance the prodifferentiation effects of h-catenin. In neural progenitor cells isolated from mouse E14 cortex, overexpression of h-catenin resulted in an increased number of neurons when the cells were allowed to differentiate by withdrawing FGF. Because the total cell number was not significantly different than control, we conclude that hcatenin promotes neuronal differentiation in the absence of other mitogenic stimuli. This finding actually correlates well with the phenotype of h-catenin transgenic mice (Chenn and Walsh, 2002). Because cells in the VZ receive local FGF signaling, the increase in h-catenin signaling in the transgenic animals would be expected to maintain cells in cell cycle and increase the size of the progenitor pool. When the cells migrate out of the VZ, however, the proneural effects of h-catenin would promote neuronal differentiation. This would lead to a preferential increase in neurons rather than all lineages as observed by Chenn and Walsh (2002). This conclusion is also consistent with prior findings regarding the effects of Wnt/h-catenin/FGF at early development stages. Inducible chimeric forms of h-catenin can induce neural markers in Xenopus embryos independent of mesoderm formation (Domingos et al., 2001). Microinjection of h-catenin mRNA also leads to

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neural differentiation in Xenopus ectodermal explants (Wessely et al., 2001). Finally, FGF2 can be used as induction agent to initiate neuronal differentiation in embryonic stem cells (Okabe et al., 1996). Target genes for the proneural effect of b-catenin How does h-catenin signaling induce neural potential in stem cells? We found that h-catenin activates the promoter or proximal enhancer of proneural genes of the bHLH family, including neurogenin 1, neurogenin 2, and to a lesser extent mash1. By DNA binding assays and luciferase reporter assays, we confirmed that there are functional Lef/ Tcf binding sites in the neurogenin promoter. Although mutation of two Lef/Tcf binding sites in the neurogenin 1 reporter inhibited its early (24 h) response to h-catenin, significant activation of this mutant reporter by h-catenin was observed at 2 days after transfection (not shown). This suggests that h-catenin may regulate neurogenin 1 through more than one mechanism. Our results from chromatin immunoprecipitation studies suggest that h-catenin binds directly to the neurogenin 1 promoter or enhancer complex both in P19 cells treated with retinoic acid and in neural stem cells in the presence of FGF2. These findings explain, at least in part, how FGF or h-catenin maintains or generates neural potential in stem cells. This conclusion is consistent with prior studies that have shown that Wnt or h-catenin signaling influences the expression of proneural genes during development. For example, Wnt1 and Wnt3a are expressed at the dorsal part of the neural tube, and in Wnt1 or Wnt3a double knockout mice, the mash1-expressing domain expands dorsally at the expense of math1 and neurogenin 1- or neurogenin 2-expressing domain (Muroyama et al., 2002). Disruption of h-catenin in neural crest blocks development of neurogenin 2-dependent sensory neurons (Hari et al., 2002). This evidence when combined with our findings supports a role for h-catenin as a general activator of proneural genes and specifically of the neurogenin genes. Although we found Lef/Tcf-dependent activation sites on the neurogenin genes, it is likely that h-catenin can also activate proneural genes through other intermediate transcription factors that are capable of regulating proneural genes activity. For example, CNS progenitors also express other genes downstream of Wnt or h-catenin, such as Iroquois 1 (Gomez-Skarmeta et al., 2001) and Pax6 (Frowein et al., 2002) that regulate proneural gene expression. Enhancing expression of proneural genes may not be the only mechanism by which Wnt/h-catenin/FGF signaling promotes neuronal differentiation. GSK-3h, a target of inhibition by both Wnt and FGF2 (Fig. 1), can inhibit the ability of neurogenin to produce ectopic N-tubulin in Xenopus without interfering with its ability to induce neuroD (Marcus et al., 1998). GSK-3h also inhibits Ntubulin expression in cultured Xenopus spinal cord (Olson et al., 1998). These results suggest that Wnt or FGF signaling may also be involved in processes downstream

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of proneural gene activation. Other mechanisms that have been proposed for explaining the neural-inducing activity of Wnt or h-catenin at different stages of development include inhibition of BMP expression at the transcriptional level (Baker et al., 1999, Gomez-Skarmeta et al., 2001) or activation of a BMP antagonist, such as Chordin (Wessely et al., 2001). In summary, our results suggest that FGF2 regulates neural stem cell proliferation and differentiation, in part, via h-catenin signaling. The proneural effects of h-catenin are suppressed under conditions that support cell proliferation such as the presence of mitogenic growth factors like FGF2. Under conditions that promote cell cycle exit, however, h-catenin promotes neuronal differentiation. This dual effect of FGF2 or h-catenin may be required for keeping neural or self-renewal potentials in neural progenitor cells both in vitro and in vivo.

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