BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells

Martin H. M. Sailer1,2,3, Thomas G. Hazel1, David M. Panchision1,4, Daniel J. Hoeppner1, Martin E. Schwab2,3 and Ronald D. G. McKay1,*

1 Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
2 Brain Research Institute, University of Zurich, 8057 Zurich, Switzerland
3 Department of Biology, Swiss Federal Institute of Technology, 8057 Zurich, Switzerland
4 Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington DC, 20010, USA

* Author for correspondence (e-mail: mckay{at}codon.nih.gov)

Accepted 21 September 2005


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
CNS stem cells are best characterized by their ability to self-renew and to generate multiple differentiated derivatives, but the effect of mitogenic signals, such as fibroblast growth factor 2 (FGF2), on the positional identity of these cells is not well understood. Here, we report that bone morphogenetic protein 2 (BMP2) induces telencephalic CNS stem cells to fates characteristic of neural crest and choroid plexus mesenchyme, a cell type of undetermined lineage in rodents. This induction occurs both in dissociated cell culture and cortical explants of embryonic day 14.5 (E14.5) embryos, but only when cells have been exposed to FGF2. Neither EGF nor IGF1 can substitute for FGF2. An early step in this response is activation of ß-catenin, a mediator of Wnt activity. The CNS stem cells first undergo an epithelial-to-mesenchymal transition and subsequently differentiate to smooth-muscle and non-CNS glia cells. Similar responses are seen with stem cells from E14.5 cortex, E18.5 cortex and adult subventricular zone, but with a progressive shift toward gliogenesis that is characteristic of normal development. These data indicate that FGF2 confers competence for dorsalization independently of its mitogenic action. This rapid and efficient induction of dorsal fates may allow identification of positional identity effectors that are co-regulated by FGF2 and BMP2.

Key words: Forebrain, Neural stem cell, Cranial neural crest, Choroid plexus mesenchyme (CPm), Epithelial-mesenchymal transition (EMT), Snai1, Snai2


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
Clonal analysis shows that stem cells can be derived from the central nervous system (CNS) and maintained in culture by the mitogenic actions of FGF2 (Johe et al., 1996Go). FGF2 and epidermal growth factor (EGF) are the only known growth factors that, by themselves, can drive the mitogenic expansion of neural precursor cells in vitro (Panchision and McKay, 2002Go). The use of FGF2 as an exogenous mitogen is supported by its actions in vivo. FGF2 is expressed together with FGF1 in the ventricular zone of the developing cortex (Dono, 2003Go; Grove and Fukuchi-Shimogori, 2003Go). Mice lacking FGF2 show a cerebral cortex size that is diminished by about 45%, affecting both neurons and glia cells (Vaccarino et al., 1999Go). However, it has also been proposed that FGFs can act as ventralizing signals in mouse cortical explants and cultured neural precursors (Gabay et al., 2003Go; Kessaris et al., 2004Go; Kuschel et al., 2003Go).

Cell fate in the developing CNS is specified in part by growth factors that are localized in distinct dorsal and ventral organizer domains (Lee and Jessell, 1999bGo; Wilson and Rubenstein, 2000Go). Bone morphogenetic proteins (BMPs) are secreted factors expressed in the prospective epidermis at the lateral edges of the neural plate, then subsequently in the dorsal midline of the neural tube (Lee and Jessell, 1999bGo; Meulemans and Bronner-Fraser, 2004Go). In the anterior neural tube, including the telencephalon, choroid plexus (CP) is the dorsal-most region. BMP signaling is both necessary and sufficient for the generation of the CP (Hebert et al., 2002Go; Panchision et al., 2001Go). In the more posterior neural tube, BMPs induce roof plate and neural crest cells (Lee and Jessell, 1999aGo). Neural crest cells are a specialized dorsal cell type, unique to vertebrates, that delaminate from the neural and/or non-neural ectoderm border from regions posterior to the mid-diencephalon, and then migrate to distant sites in the embryo (Bronner-Fraser, 2002Go; Knecht and Bronner-Fraser, 2002Go; Trainor et al., 2002Go; Wu et al., 2003Go). The neural crest gives rise to peripheral nervous system (PNS) derivatives such as peripheral neurons and Schwann cells, along with non-neural derivatives such as melanocytes, craniofacial chondrocytes, osteocytes and perivascular cells (smooth muscle, pericytes, connective tissue) (Gammill and Bronner-Fraser, 2003Go; Le Douarin and Kalcheim, 1999Go; Meulemans and Bronner-Fraser, 2004Go; Trainor et al., 2002Go; Wu et al., 2003Go).

Previous work has shown that BMPs promote the generation of mesenchymal derivatives with neural-crest-like phenotypes from CNS precursors in vitro (Gajavelli et al., 2004Go; Mujtaba et al., 1998Go; Rajan et al., 2003Go). This is in contrast to other studies that have shown CNS neuronal or glial differentiation after BMP treatment (Gross et al., 1996Go; Li et al., 1998Go; Mehler et al., 2000Go), suggesting a context-dependent component to the BMP response. In this study, we show that exposure to FGF2 is required for BMPs to generate neural-crest-like cells in cortical explants or cultured stem cells. Co-treatment with FGF2 and BMP2 rapidly upregulates ß-catenin, a mediator of Wnt activity (Moon et al., 2004Go) and of Bmp2 itself, suggesting a positive feedback of BMP-signaling. This is consistent with a role for Wnt-signaling in regulating dorso-ventral identity in the developing telencephalon of chick and mouse (Backman et al., 2005Go; Gunhaga et al., 2003Go). FGF2 and BMP2 treatment initially induces genes associated with the epithelial-mesenchymal transition (EMT) to a neural-crest-like state (Nieto, 2002Go), followed by terminal differentiation into derivatives such as smooth muscle and non-CNS glia. Forebrain stem cells from multiple ages retain the competence for this dorsal respecification. Peripheral neurons are not generated under these conditions, but can be induced at low frequency when stem cells are co-treated with the posteriorizing factor retinoic acid. Thus, although these cell types are characteristic of neural crest, the anterior origin of the responding cells biases the FGF-BMP-Wnt-induced dorsal transition to a phenotype most similar to cranial mesenchyme or choroid plexus mesenchyme (CPm).


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
Growth factors
Growth factors were used at the following concentrations (all from R&D Systems with BSA as carrier protein, if not otherwise stated): recombinant human (rh) FGF2 at 10 ng/ml (146 aa) for fetal and at 20 ng/ml for adult stem cells; rhBMP2, BMP4, BMP7 at 20 ng/ml; recombinant mouse noggin at 2.5 ng/ml; rhIGF1 at 330 ng/ml; bovine pancreatic insulin at 25 µg/ml (Sigma). To promote neuronal survival and maturation, we used B27 supplement (1:100 dilution, Invitrogen), 40 nM retinoic acid (Sigma), 10 ng/ml nerve growth factor (rhNGF), 10 ng/ml heregulin ß1 (rhHRG), 10 ng/ml recombinant rat brain-derived growth factor (BDNF) and 10 ng/ml glial-cell-line-derived neurotrophic factor (rrGDNF).



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Fig. 1. Expression of p75NGFR, SMA and GFAP in the E14.5 rat forebrain and cranial mesenchyme. Fluorescent antibody staining of E14.5 rat embryo at the telencephalic level. Illustration at upper left orients panels A-G. Red rectangle with asterisk illustrates the cortical region dissected for all experiments. (A) p75NGFR (green) and smooth muscle {alpha}-actin (red) show little or no expression in brain but high levels of p75NGFR in cranial mesenchyme and peripheral ganglia. Arrow indicates choroid plexus. (B-D) Higher magnification, showing (B) cranial mesenchyme, (C) choroid plexus (arrow) and confocal image of choroid plexus epithelium (CPe) and (D-F) mesenchyme. Notice that some CPm cells are p75NGFR+/SMA+, whereas the CPe shows fainter staining for these markers. (G) CPe shows moderate co-expression of p75NGFR (green) and GFAP (red), whereas the CPm is strongly p75NGFR+/GFAP+. Cranial mesenchyme is stongly p75NGFR+ but only weakly GFAP+. DAPI (blue) identifies all cell nuclei. Bars, 160 µm (A); 40 µm (B-C, E); 15 µm (D).

 
Culture of cortical explants
E14.5 rat telencephalon was dissected to be completely free of meninges. A cortex section of about 800 µm by 3200 µm along the length of the medial ganglionic eminence (MGE) in a distance of about 800 µm from the MGE and starting at the anterior MGE pole was dissected (Fig. 1). This section was used for explants of 400-800 µm in diameter. The explants were generated by cutting the cortex tissue section into smaller pieces with a microsurgical needle (tungsten); they were grown in the same conditions as CNS stem cells (see below), except for the omission of insulin, progesterone, putrescine and selenium from the medium (basal medium). Transferrin, an iron-binding protein necessary for efficient iron metabolism, was maintained in culture because its removal caused growth retardation and some cell death (data not shown). Medium with growth factors was replaced every other day. The explants were washed twice with basal medium between the first and second phase of treatment (see Table 1). The responses to growth factors were recorded by phase-contrast microscopy using a Zeiss Axiovert 10 microscope (Carl Zeiss Inc.).


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Table 1. p75NGFR induction in E14.5 cortical explants

 

Culture of CNS stem cells
Both fetal and adult CNS stem cells were isolated and cultured as previously described (Johe et al., 1996Go; Kim et al., 2003Go) in Dulbecco's modifies Eagle's medium (DMEM) F12 with N2 supplements (Kim et al., 2003Go), unless otherwise noted. We used E14.5- (Taconic E15) and E18.5- (Taconic E19) timed pregnant Sprague Dawley rats (Taconic, Germantown, NY; noon of day of plug detection equals E0.5). Adult rats (8 to 12 weeks old, Sprague Dawley, Taconic) were deeply anesthetized in methoxyflurane (Mallinckrodt, Mundelein, IL) before decapitation and dissection of the subventricular zone. For adult cultures, noggin was added during FGF2 expansion because we detected low levels of BMP expression in these cells, consistent with previous results (Lim et al., 2000Go). Cultures were maintained in this manner for 5 days; noggin was withdrawn prior to initiating experiments.

Experiments were performed upon passage 1 or 2 using 10 ng/ml FGF2 and 20 ng/ml BMP2 unless otherwise noted. Cells were cultured at low density (38 cells/cm2) for clonal analysis, medium density (380 cells/cm2) to generate distinct colonies, or high density (2546 cells/cm2), all based on previous studies (Rajan et al., 2003Go). To assay early precursor induction, cells were treated with FGF2 ± BMP2 for varying durations prior to cell fixation or harvesting. To assay differentiated cell types, one treatment paradigm involved 1 day of FGF2 expansion, then 2 days FGF2 ± BMP, then 5 days mitogen withdrawal ± BMP2 to maximize post-mitotic cell maturation. A second BMP2 treatment paradigm involved 8 days of BMP2 treatment, in varying doses or transient exposure, during continuous FGF2 expansion prior to fixation.

Immunocytochemistry
Embryo explants or cultured cells were fixed with freshly made, cold paraformalehyde (4%) and processed as described (Panchision et al., 2001Go). Cells were stained with primary antibodies against the following proteins: Brn3a (rabbit, 1:2000, gift of Eric Turner), calponin (mouse, 1:500, Sigma), ß-catenin (mouse, 1:50, Upstate), galactocerebroside (GalC, mouse, 1:200, Chemicon), glial fibrillary acidic protein (GFAP) (rabbit, 1:600, Dako), nestin (rabbit serum 130, 1:50 dilution, R.D.G.M.'s laboratory), p75NGFR (mouse, 1:100, Calbiochem), p75NGFR (rabbit, 1:200, Chemicon), protein zero (P0, mouse, 1:200, gift of Juan Archelos), smooth-muscle {alpha}-actin (SMA) (mouse, 1:800, Sigma), smooth-muscle myosin heavy-chain 1+2 (SMMHC, rabbit, 1:800, gift of Robert Adelstein), Sox9 (rabbit, 1:50, Chemicon), peripherin (mouse, 1:100, Chemicon; rabbit, 1:2000, Chemicon), tyrosine hydroxylase (mouse, 1:1000, Sigma; rabbit 1:400, PelFreez). For ß-catenin staining, cells were fixed in 4% PFA containing 4% sucrose at 4°C for 5 minutes, then in 100% methanol at –20°C for 15 minutes and then extensively washed in PBS. Appropriate secondary antibodies (Cy3, 1:300, Jackson Immunoresearch, or Alexa Fluor 488, 1:200, Alexa Fluor 555, 1:300, Molecular Probes) were applied and incubated for 1 hour at room temperature. Nuclei were labeled with 0.25 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 15 minutes. Images were photographed, using fluorescent filters, with a Zeiss Axioplan microscope and Zeiss Axiocam HR camera (Carl Zeis Inc.) or a Zeiss confocal microscope (LSM 510). Statistical analysis and histogram illustration of cell numbers were performed using SigmaPlot 5.0 (SPSS Inc.) and Prism 4.0 (Graphpad Software Inc.). All images were combined for figures using Photoshop 7.0 for Windows (Adobe).

Western blotting
Protein was harvested as described before (Rajan et al., 2003Go) using protease and phosphatase inhibitors. Standardized protein was loaded on 4-12% polyacrylamide gels and blotted on nitrocellulose membranes (both Invitrogen); a positive control (Upstate) was included. A non-phosphorylated 12-amino-acid (aa) peptide corresponding to aa 36-44 in human ß-catenin was used as the immunogen for the activated ß-catenin antibody. Blots were probed with HRP-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch) and the antibodies reacted with a chemiluminescent reagent (1:2, Pierce).

Reverse-transcription PCR analysis
For gene expression analysis, cultures were harvested in Trizol reagent (Invitrogen) for isolation of RNA and treated with DNaseI for 15 minutes at room temperature. The RNA (1000 ng in fetal, 300 ng in adult cell cultures) was reverse-transcribed into first-strand cDNA using oligonucleotide (dT)-primers at 0.025 µg/µl in a 40 µl reaction (Invitrogen). A 0.5 µl aliquot of the first strand reaction in a total volume of 30 µl was used for PCR. The following intron-spanning primers were used: Snail1 (rat, accession no. XM_342587, nucleotides 423-438 and 730-715, 307 bp product), 1.5 mM MgCl2, 57°C annealing; fetal cells: 39 cycles, adult cells: 40 cycles; Snail2 (rat, accession no. AF497973, nucleotides 291-313 and BC062164, nucleotides 931-915, 522 bp product), 1.5 mM MgCl2, 57°C annealing, fetal cells: 34 cycles, adult cells: 34 cycles; Bmp2 (rat, accession no. Z25868, nucleotides 222-242 and 684-662, 463 bp product), 1.5 mM MgCl2, 57°C, 40 cycles; GAPDH (rat, accession no. AF106860, nucleotides 687-704 and 988-970, 301 bp product), 1.5 mM MgCl2, 57°C annealing, fetal cells: 28 cycles, adult cells: 31 cycles. Products were sequence-verified.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
Expression of p75NGFR, SMA and GFAP in the E14.5 rat forebrain and cranial mesenchyme
Previous results from our lab (Panchision et al., 2001Go; Rajan et al., 2003Go; Tsai and McKay, 2000Go) and others (Gajavelli et al., 2004Go; Mujtaba et al., 1998Go) indicate that cells with neural crest characteristics can be generated from CNS tissue, particularly in response to BMP treatment. These cells are distinguished by the early expression of the low-affinity NGF receptor (NGFR) (p75NGFR), a transient marker of multipotent neural crest stem cells and some derivatives (Morrison et al., 1999Go), and the subsequent expression of markers for either smooth muscle (SMA) or glia (GFAP) (Gajavelli et al., 2004Go; Mujtaba et al., 1998Go; Rajan et al., 2003Go; Tsai and McKay, 2000Go). Interestingly, this in-vitro response appears with CNS precursor cells that are developmentally older than the ages (E8.5-9.5) at which neural crest is normally generated (Serbedzija et al., 1992Go), suggesting that these CNS cells possess a latent capacity to generate non-CNS fates. Since BMP signaling in vivo is both necessary and sufficient for generation of CP, the dorsal-most telencephalic identity (Hebert et al., 2002Go; Panchision et al., 2001Go), an alternative possibility is that these cells are exhibiting properties of CP rather than neural crest.



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Fig. 2. FGF2 and BMP2 induce CNS stem cells to neural-crest-like precursor state. (A) RT-PCR analysis of Msx1 expression in passage 1 of medium-density-plated stem cells treated with FGF2 or FGF2 and BMP2 after 3 days. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. (B) RT-PCR analysis of Snail1 and Snail2 expression under same conditions over 7 days. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. (C) Phase images of FGF2- or FGF2 and BMP2-treated E14.5 rat cortical stem cell cultures at medium density. Notice the initial extension of reticulated processes and cell flattening during BMP2 treatment. (D-G) Comparison of p75NGFR expression in passage 1 cells plated at clonal density; clones marked after 4 days FGF2 expansion (7.7±1.7 cells/clone) were further expanded ±20 ng/ml BMP2 treatment. By 5 days, p75NGFR expression is absent in (D) FGF2-expanded cells but prevalent in (E) FGF2 plus BMP2-treated cells. Percent clones containing any (F) p75NGFR+ cells and total percentage of (G) p75NGFR+ cells during FGF2 expansion without ({square}) or with ({diamondsuit}) BMP2. Graphs show the mean ± s.e.m. (n=3). Bars, 80 µm (C); 40 µm (D,E).

 
To identify forebrain cells expressing these markers, the normal expression pattern of p75NGFR and SMA in the E14.5 rat head was defined. The cranial mesenchyme and CP mesenchyme (CPm) both expressed p75NGFR (Fig. 1A-C). The cranial mesenchyme derives from neural crest cells that migrate from regions caudal to the mid-diencephalon (Le Douarin and Kalcheim, 1999Go); the CPm is adjacent to the choroid plexus epithelium (CPe) and is of undetermined origin in rodents. The CPe also expressed p75NGFR but at lower levels than in the CPm (Fig. 1D). As expected, p75NGFR expression was prominent in other neural crest structures such as peripheral ganglia. By contrast, SMA expression was localized to a subset of p75NGFR-positive (p75NGFR+) cells in the head mesenchyme in vascular-like structures intimately associated with the CPe (Fig. 1D-F). Confocal microscopy confirmed the presence of p75NGFR +/SMA+ cells in the CPm (Fig. 1D-F).

GFAP expression was minimal in the E14.5 rat forebrain, consistent with the low frequency of glial differentiation at this early age. CPe expressed GFAP at low levels, but stronger than that seen in more ventral neural epithelium; there were also weakly co-expressing GFAP+/p75NGFR+ cells (Fig. 1G). GFAP expression was higher in the cranial mesenchyme, including cells in the CPm that were GFAP+/p75NGFR+. Coexpression of GFAP and p75NGFR is characteristic of non-myelinating Schwann cells (Zorick and Lemke, 1996Go; Zorick et al., 1996Go). Thus, CPm cells display characteristics that are between CPe and neural crest derivatives, such as cranial mesenchyme.

BMP2 and FGF2 efficiently induce CNS stem cells to a P75NGFR+/SNAIL+ neural-crest-like precursor state
We next measured the timing and efficiency of CNS stem cell responses to BMPs. These cells normally give rise to neurons, astrocytes and oligodendrocytes that are characteristic of their tissue of origin (Johe et al., 1996Go; Vicario-Abejon et al., 2000Go). Dissociated cells from E14.5 rat cortex were expanded in FGF2 for 4 days and then passaged and plated at medium density (for details see Materials and Methods) just before BMP treatment. Co-treatment with BMP2 and FGF2 induced the expression of Msx1 (Fig. 2A), which is expressed in dorsal midline cells including CPe, and of Snail1 and Snail2 (formerly known as Snail1 and Slug, respectively) (Fig. 2B), both of which identify CPm and neural crest precursors and are required for proper epithelial to mesenchymal transition (EMT) to a migratory phenotype (Aybar et al., 2003Go; Etchevers et al., 2002Go; Marin and Nieto, 2004Go; Nieto, 2002Go). This co-treatment caused pronounced morphological changes in the cells, including the formation of reticulated processes and eventual cell flattening by 6 days (Fig. 2C). FGF2-BMP2 co-treatment also induced p75NGFR, a surface receptor expressed in CPm and neural crest precursors (Etchevers et al., 2002Go). By clonal analysis, we found that BMP2 co-treatment generated p75NGFR+ cells in every clone within 4 days (Fig. 2D-F), indicating that all initially plated and FGF2-expanded fetal stem cells were responsive to BMP2. The expression of p75NGFR commenced within 2 days of BMP2 treatment and reached a maximum frequency of about 60% of all cells after 5 days BMP2 treatment (Fig. 2G). By this time, the p75NGFR-negative (p75NGFR–) cells also had a similarly flattened morphology, indicating that they also were BMP2-responsive (data not shown).



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Fig. 3. FGF2 is required for BMP2-mediated EMT. Analysis of p75NGFR expression in E14.5 rat cortical explants cultured in basal medium (DMEM-F12 without N2) alone or supplemented with growth factors (see Table 1). Treatments include a 4-day first phase and a 5-day second phase. Low-magnification images show p75NGFR cytoplasmic staining (green) with DAPI+ nuclei (blue); original explants are shown on the right edge of each image. Only media containing FGF2+BMP2 (I-L, P) were able to induce neural crest precursors as measured by p75NGFR expression and migration from explant. Neither insulin nor IGF1 was required (I-K) and IGF was not sufficient (B,D,G) to induce p75NGFR+ precursors. (M,N) Substitution of EGF for FGF2 was not sufficient for neural crest induction. (O,P) A small number of p75NGFR+ cells with thin processes in three of 42 IGF1/IGF1+BMP2 treated explants (O) that differ from the flattened morphologies of FGF2+BMP2/FGF2+BMP2 treated explants (P). (Q) RT-PCR panel showing Snail2 induction only in explants cultured with both FGF2+BMP2. Abbreviations: F, FGF2; I, IGF1; E, EGF; B, BMP2. Factor concentrations listed in Materials and Methods. Bars, 80 µm (A-M); 80 µM (N-P).

 
BMP4 treatment has been shown to induce cell death in explants of E10.5 mouse cortical tissue (Furuta et al., 1997Go), raising the possibility that the pronounced inductive response we saw does not reflect the potential of most cortical stem cells. No detectable cell death in cortical stem cell cultures was observed during the first four days of BMP4 treatment, as measured by pyknotic cell counting and fragmented nuclei or staining for activated caspase-3 (data not shown). The fact that BMP2 treatment induced Snail1 and Snail2 expression and that all clones contained p75NGFR+ cells after 4 days of treatment is consistent with an efficient dorsal induction of CNS stem cells to a mesenchymal cell type such as neural crest or CPm (Marin and Nieto, 2004Go).

FGF2 is required for BMP2-mediated epithelial-to-mesenchymal transition (EMT)
To determine whether the response of cultured stem cells to BMP2 reflects an EMT, we dissected the same cortical tissue as for our dissociated cultures but instead cultured the whole explants of 400-800 µm in diameter. Similar delamination and migration paradigms using early-gestation neural tube explants have been exploited to prepare neural crest cultures (Kleber et al., 2005Go; Lee et al., 2004Go). The explants were cultured in a `basal medium' (DMEM-F12 plus transferrin) plus different combinations of additional factors (Table 1). The explants were first cultured under conditions to manipulate CNS-cell-expansion (Phase I, 4 days), and then under conditions to test EMT (phase II, 5 days), after which the explants were fixed and stained for p75NGFR.

The basal medium by itself did not support cell migration or the induction of p75NGFR (Table 1, experiment A). We then tested the effect of IGF1 instead of insulin, because IGF1 has a tenfold stronger survival-promoting activity than insulin in CNS stem cells at the same concentration (Drago et al., 1991Go). FGF2-IGF1 co-treatment caused an elaboration of cells from the explant with a characteristic CNS-precursor morphology (Fig. 3D) and did not induce p75NGFR expression (Table 1, experiments B-D). The high density of DAPI+ cells suggested that this was an overgrowth of CNS precursors. FGF2 or BMP2 alone were not sufficient to induce p75NGFR or migration (Table 1, experiments E, L, O). Culture of explants with either FGF2 or FGF2-IGF1 in phase I, followed by BMP2 exposure in phase II, strongly induced p75NGFR expression (Fig. 3I-L, Table 1 experiments F and H) and Snail2 expression (Fig. 3Q). A shorter 2-day exposure of cortical explants also induced p75NGFR (Fig. 3J, Table 1, experiment Q). The p75NGFR+ cells migrated away from the explant and adhered tightly to the surface of the well; this migratory effect was not seen in cultures that did not generate p75NGFR+ cells. Furthermore, SMA+ cells were found exclusively in these migratory regions (not shown).

By contrast, treatment with IGF1 alone, followed by IGF1 plus BMP2 was only able to induce a total of seven p75NGFR+ cells in three out of 42 cortical explants (Fig. 3G,O; Table 1, experiment G); these cells were small and elongated compared to the large flat p75NGFR+ cells of the FGF2-BMP2-treatments (Fig. 3P, Table 1, experiments F and H). Additionally, no Snail1/2 expression was seen in IGF1-BMP2 cultures (Fig. 3Q), excluding the possibility that they are CPm or neural crest precursors. Cells expressing p75NGFR were not seen in explants exposed to EGF, the other known mitogen for cultured CNS stem cells (Table 1, experiments I and J). In contrast to previously reported experiments carried out in chick explants at an earlier developmental time point (Garcia-Castro et al., 2002Go), the combination of N2 and BMP2 was not sufficient to induce an EMT in rat cortical explants (Table 1N,O). Furthermore, only the combination of FGF2 and BMP2 induced the expression of Snail2 (Fig. 3Q). These experiments indicate that the functions of both BMP2 and FGF2 are required to induce a migratory cell population that expresses genes characteristic of CPm or neural crest.



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Fig. 4. BMP2-mediated activation of a Wnt signal and Bmp2 expression. E14.5 rat cortical stem cells were plated at 5092 cells/cm2 and exposed to 20 ng/ml BMP2 for 0, 24 or 48 hours. (A) Activated ß-catenin levels increased after 24 and 48 hours, as shown by western blotting. Activated ß-catenin control-cell lysate and {alpha}-tubulin were included as references. (B-E) Immunocytochemistry, showing increased ß-catenin activation after 24 hours of BMP2 treatment; control cells show only few faintly positive cells; (B,D) DAPI staining indicates total cell nuclei. (F) RT-PCR time course analysis of medium-density cultures after exposure to FGF2 or FGF2 and BMP2. One day of BMP2 exposure was sufficient to upregulate transcription of endogenous Bmp2 mRNA. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) samples as loading controls. Bars, 20 µm.

 

    BMP2-mediated activation of a Wnt signal and BMP2 expression
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
Secreted proteins of the Wnt family have been implicated in the dorsalization of the telencephalon and in the induction and maintenance of neural crest (Backman et al., 2005Go; Garcia-Castro et al., 2002Go; Gunhaga et al., 2003Go; Kleber et al., 2005Go; Saint-Jeannet et al., 1997Go). Activation of the canonical Wnt pathway leads to translocation of ß-catenin to the nucleus and to subsequent transcriptional activation in conjunction with members of the T-cell factor/lymphocyte enhancer binding factor (TCL/LEF) family (Moon et al., 2004Go). Nuclear ß-catenin localization was used as a measure for activated canonical Wnt-signaling in FGF2-expanded and BMP2-treated cortical stem cells. FGF2-expanded stem cells express low levels of nuclear ß-catenin (Fig. 4A,B). BMP2 exposure increased levels of nuclear ß-catenin signal within 24 hours (Fig. 4C-E). This indicates that BMP-stimulation rapidly activates the canonical Wnt-signaling pathway in NSCs. Since BMP signaling is tightly regulated during neural development, Bmp2 mRNA levels were also measured. Bmp2 mRNA was not detectable in control cultures, but was upregulated within 24 hours of BMP exposure (Fig. 4F). During continuous BMP exposure the Bmp2 mRNA remained elevated (Fig. 4). Furthermore, transient exposure to BMP2 for 2 days was sufficient to induce p75NGFR 7 days later (Table 1Q). Thus, initial BMP exposure might activate a positive feedback loop of BMP signaling.


    FGF2 and BMP2 differentiate CNS stem cells to multiple non-CNS derivatives
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
Even in the continued presence of FGF2, BMP2-treated cells eventually stopped proliferating and most adopted a flat morphology. In low-density cultures, BMP treatment caused efficient differentiation to cells expressing smooth-muscle {alpha}-actin (Fig. 5), which is consistent with our previous results (Rajan et al., 2003Go). This response was specific for the BMP subclass of transforming growth factor ß (TGFß) factors. Neither FGF2 withdrawal, which is known to induce differentiation in neural stem cells, nor 8 ng/ml TGFß1, which is known to promote smooth-muscle differentiation in neural crest stem cells (Shah et al., 1996Go), induced any SMA+ cells (Fig. 5B-C, M). By contrast, BMP2, BMP4 or BMP7 induced SMA expression in 92.8%, 96.6% or 90.6% of all cells, respectively, when treated with concentrations of 20 ng/ml (Fig. 5D-F,M).



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Fig. 5. BMP-treated CNS stem cells efficiently differentiate into smooth muscle and co-express Sox9, SMMHC1+2 and calponin. (A) Differentiation paradigm 1 (for panels B-F,M) and paradigm 2 (panels G-L,N-V). Cortical stem cells were seeded at low density after first passage. (B-F) Cells differentiated by (B) FGF2-withdrawal or (C) 8 ng/ml TGFß1 co-treatment are all SMA. By contrast, 20 ng/ml of BMP2, BMP 4 or BMP 7 efficiently generate SMA+ cells. (M) Quantitation of B-F. Notice that the passage 5 experiment varies from paradigm 1 by using a 7-day co-treatment and 7-day withdrawal. (G-L) Dose-response assay showing no or few SMA+ cells in (G) FGF2-expanded or (H,I) low-dose BMP2 co-treated cells. (J) Percentage of SMA+ cells increases at 10 ng/ml BMP2 but yields immature-looking cells, and plateaus to almost 100% at 20 ng/ml BMP2, even during continued mitogenic expansion (K,L, quantitation in N). (O) Initial transient exposure to 20 ng/ml BMP2 during FGF2 expansion is sufficient to induce SMA+ differentiation by 8 days. (P-V) Cells co-treated with 10 ng/ml FGF2 (F) and 20 ng/ml BMP2 (B) nearly all co-express SMA and Sox9 (Q-R), SMMHC (S-T) and calponin (U-V). Graphs show mean ± s.e.m. (n=3). Bars 40 µm (B-L); 20 µm (Q-V).

 
The myogenic potential of cortical stem cells was maintained over five passages in culture, corresponding to more than 4 weeks in expansion (Fig. 5M). A dose-dependent effect of BMP2 on differentiation to SMA+ cells was found when cortical stem cells were co-treated with FGF2 for 8 days. Low doses of BMP2 generated few SMA+ cells, whereas 10 ng/ml BMP2 yielded larger numbers of SMA+ cells having a small size and immature morphology (Fig. 5G-J,N). Both the frequency and maturity of these cells increased in conditions where FGF2 was withdrawn after the initial co-treatment with BMP2 (data not shown). At doses of 20 ng/ml or higher, the cells assumed a typical sheet-like morphology containing parallel stress fibers (Fig. 5K-L,N). Even transient exposure to 20 ng/ml BMP2 was sufficient to direct cells to a smooth-muscle fate (Fig. 5O).

After treatment with 20 ng/ml BMP2, nearly all cells were positive for SMA, calponin and smooth-muscle myosin heavy-chain (SMMHC) 1 and 2 (Fig. 5P,S-V). These cells also showed nuclear expression of Sox9 (Fig. 5P-R) and p21cip1 (data not shown) (Panchision et al., 2001Go), consistent with their identity as post-mitotic smooth-muscle cells. This expression was not seen in untreated cells nor TGFß1-treated cells (Fig. 5Q,S,U and data not shown).

Apart from smooth-muscle cells, BMP2-treated CNS stem cells also differentiate to GFAP+ glia (Rajan et al., 2003Go). In individual colonies, SMA+ cells predominated at the sparse edges, whereas the proportion of GFAP+ cells increased dramatically in the dense core (supplementary material, Fig. S1), consistent with our previous results (Rajan et al., 2003Go). Varying the density before treatment with BMP yielded almost homogeneous populations of either SMA+ or GFAP+ cells. To further characterize the glia cells, high-density cultures treated with FGF2 and BMP2 were stained for both GFAP and p75NGFR. A majority of cells were GFAP+/p75NGFR+, consistent with a non-myelinating Schwann cell fate (Fig. 6A-C) (Zorick and Lemke, 1996Go). A low proportion (0.7%) co-expressed galactocerebroside (GalC) and p75NGFR (Fig. 6D-F), which together mark both pro-myelinating and non-myelinating Schwann cells (Jessen and Mirsky, 2002Go; Zorick et al., 1996Go). This distinguishes them from oligodendrocytes of the intact adult brain that do not express p75NGFR under normal, non-injured circumstances (Beattie et al., 2002Go). Myelin protein zero (MYP0), another gene expressed at high levels in mature Schwann cells (Jessen and Mirsky, 2002Go), was also detectable at low levels in our cultures by immunohistochemistry and RT-PCR (data not shown). MYP0 protein was found in a high proportion of cells but at low levels (data not shown), similar to immature neural crest cells (Hagedorn et al., 1999Go). These cells exhibit characteristics indicating a non-myelinating, non-CNS glia identity and are also consistent with the cell types seen in the CPm.



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Fig. 6. BMP-treated CNS stem cells differentiate to non-myelinating, non-CNS glia. (A-F) Generation of glia cells using paradigm 1 after high-density plating. (A-C) Control cultures generate immature GFAP+ (green) astrocytes (A), whereas BMP2 co-treatment yields distinctively flattened cells, most co-expressing GFAP (green) and p75NGFR (red), consistent with a non-myelinating Schwann cell phenotype (B, quantitation in C). (D-F) Control cultures generate only small numbers of GalC+ CNS oligodendrocytes and no GalC+/p75NGFR+ cells (D), wheras BMP2 co-treatment yields morphologically distinct GalC+ (red) and 75NGFR+ (green) co-expressing cells (D, quantitation in F). (G-I) Peripherin+ neurons could be generated in acute cultures only by adding retinoic acid during a 3-day FGF2/BMP2 co-treatment, followed by BDNF, GDNF, NGF and HRG during a 7-day mitogen withdrawal. Peripherin+ cells had long and sometimes branched processes (G, quantitation in I); 92% of peripherin+ cells co-expressed Brn3a (H), consistent with a peripheral neuron identity. Graphs show mean ± s.e.m. (n=3-4). Bars, 20 µm (A,B); 10 µm (D,E); 80 µm (G), 10 µm (H).

 

BMP-exposure did not reproducibly generate cells that expressed peripheral neuronal markers. We addressed this by adding retinoic acid, a known posteriorizing factor in neural crest induction (Villanueva et al., 2002Go), during the FGF2-BMP2 co-treatment in acutely dissociated cells. We then included B27, NGF, Heregulin-ß1, BDNF and GDNF along with BMP2 during the FGF2-withdrawal phase. We observed cells expressing peripherin (Fig. 6G-I), an intermediate filament found in peripheral neurons and some population of CNS neurons projecting into the periphery (e.g. cranial nerve ganglia and motoneurons), but also in rare neuronal populations of the neocortex (Rhrich-Haddout et al., 1997Go). Nearly all (92%) of the peripherin+ cells co-stained for Brn3a (Brn3.0, Fig. 6H), a POU transcription factor that is expressed in nearly all dorsal root sensory neurons (Anderson, 1999Go), but not in the cortex. We could not reproducibly find cells expressing tyrosine hydoxylase (TH) (data not shown), an enzyme expressed in peripheral autonomic neurons. Thus, under the appropriate posteriorizing conditions we were able to identify cells consistent with a peripheral neuronal identity, although the absence of sufficient trophic factors might account for the low frequency. We were unable to identify other neural crest derivatives such as melanoblasts, melanocytes or chondroblasts as measured by Kit, MITF, Trp2 or collagen 2{alpha}1 protein and Trp2 and collagen 2{alpha}1 mRNA (not shown).

In contrast to FGF2-BMP2 co-treated cells, the cultures grown only in FGF2 (with or without retinoic acid) did not generate peripherin+ cells (Fig. 6I), GFAP+/p75NGFR+ cells (Fig. 6C) or GalC+/p75NGFR+ cells (Fig. 6F), nor cells staining for more mature Schwann cell markers such as MAG, MBP or MYP0 (data not shown). Thus, the generation of these non-CNS differentiated derivatives depended on the specific actions of BMPs.


    Both fetal and adult CNS stem cells generate dorsalized derivatives
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
The data from E14.5 cortical cells showed that FGF2 conferred a competence to generate mesenchymal derivatives similar to CPm or neural crest in response to BMPs. Adult CNS stem cells are similar to fetal stem cells in their responses to factors that control neuronal and glial differentiation (Johe et al., 1996Go), indicating that they share common signaling mechanisms. BMP2 and BMP4 are expressed in the adult mouse subventricular zone (SVZ) and their gliogenic actions on multipotent SVZ cells are antagonized by the secreted factor noggin (Lim et al., 2000Go). We added noggin to our SVZ cultures during expansion to minimize any confounding effect of endogenous BMPs. After the first passage, we tested adult SVZ cultures for mesenchymal induction.



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Fig. 7. FGF2 and BMP2 induce adult rat SVZ stem cells to dorsalized precursors. Adult SVZ stem cells were expanded in FGF2 with noggin prior to passage. (A) RT-PCR time course analysis of Snail1 and Snail2 expression in passage 1, during FGF2 ± BMP2 exposure. GAPDH expression is shown in reverse-transcribed (+) and non-transcribed (–) GAPDH samples as loading controls. (B-E) Comparison of p75NGFR expression after low-density plating; clones marked after 5 days expansion (116±54 cells/clone) were further expanded with FGF2 ± BMP2. Expression of p75NGFR is absent in (B) control cultures but prevalent in (C) BMP2 co-treated cells. Percentage of clones containing (D) at least one p75NGFR+ cell and total percentage of (E) p75NGFR+ cells during FGF2 expansion without ({square}) or with ({diamondsuit}) BMP2. Graphs show mean ± s.e.m. (n=2). Bars 20 µm.

 
BMP2 treatment upregulated the expression of Snail1 and Snail2 beginning at day 1 and continuing through 5 days of treatment as measured by RT-PCR (Fig. 7A). The neural crest markers AP-2, noelin-1 and foxd3 were induced after 4 hours of treatment, whereas the peripheral glia marker MYP0 was induced after 5 days (not shown). In clonal analysis, no p75NGFR+ cells were seen in FGF2-expanded cells (Fig. 7B), whereas addition of BMP2 led to the induction of p75NGFR+, starting at day 1 and peaking at day 3 of the treatment (Fig. 7C-E). Expression of p75NGFR occurred faster in adult stem cells than in the fetal stem cells; almost all cell clusters contained p75NGFR+ cells after 2 days of BMP2 co-treatment (Fig. 7D). In contrast to the fetal cultures, the maximum frequency of p75NGFR expression in adult cultures was only 30% at day 3 and decreased thereafter (Fig. 7E), consistent with the idea that p75NGFR expression often marks a transient precursor state (Morrison et al., 1999Go).

Whereas control cultures generated no SMA+ cells (Fig. 8A), FGF2/BMP2 co-treatment generated cells that expressed both SMA (8.1±2.7%, Fig. 8B,C) and calponin (8.8±3.7%). Interestingly, the proportion of smooth-muscle cells (SMA+) induced by BMP exposure decreased, depending on the isolation age. When controlling for culture conditions, the percentage of SMA+ cells progressively decreased from E14.5 to E18.5 to adult cells (Fig. 8C).



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Fig. 8. BMP-treated adult SVZ stem cells preferentially differentiate into non-CNS glia. Passaged adult SVZ stem cells were plated at low density, FGF2-expanded for 3 days and then treated with FGF2 ± BMP2 for 7 days. (A) Control cultures generate no SMA+ cells; (B) BMP2 co-treatment yields flattened SMA+ cells; (C) Percent SMA+ cells is decreased in cells from older embyos and adults. (D) Control cultures generate thin, immature GFAP+ cells; (E) BMP2 co-treatment yields flattened GFAP+ cells; (F) Percent GFAP+ cells increases as a function of animal age (F). (G-I) FGF2-expanded cultures and cultures where FGF2 was withdrawn generate infrequent GalC+ cells with immature CNS oligodendrocyte morphologies and do not contain p75NGFR+ cells (G), whereas BMP2 co-treatment yields flattened p75NGFR+ cells, some of which co-express GalC (H). The percentage of FGF2-BMP2-generated GalC+/p75NGFR+ cells increases as a function of animal age (I). All graphs show mean ± s.e.m., n=3-4. Bar, 20 µm.

 
By contrast, adult cells generated larger numbers of glia cells in response to BMP2. Whereas differentiated control cultures contained weakly stained GFAP+ cells with an immature astrocytic morphology (Fig. 8D), BMP2 co-treatment generated large numbers of distinctly flattened, strongly-expressing GFAP+ cells (Fig. 8E), most of which co-expressed p75NGFR (not shown). The proportion of total GFAP+ cells increased with the age of the donor animal (Fig. 8F). A smaller proportion of BMP2-treated adult cells were GalC+/p75NGFR+ (5.8±3.3%) although this also represented an increase over that seen in fetal cultures (Fig. 8G-I). Since EGF is a commonly used mitogen for adult SVZ stem cells, we tested EGF-BMP2 co-treatment and found neither p75NGFR+ nor SMA+ cells in these cultures (data not shown). Thus, adult SVZ cells also retain the capacity to be dorsalized to CPm or neural crest derivatives in response to BMP2 and also require FGF2 for this action. Whereas BMP-mediated dorsalization occurs in CNS stem cells from different ages, the propensity toward glial differentiation is higher in adult compared with fetal CNS stem cells.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 
The neural crest contains multipotent cells that can acquire PNS and other fates (Baroffio et al., 1988Go; Bronner-Fraser and Fraser, 1988Go). Many studies have identified roles for FGF, BMP and Wnt signals in the induction of neural crest from the neural-plate-stage embryo (Aybar et al., 2002Go; Garcia-Castro et al., 2002Go; LaBonne and Bronner-Fraser, 1998Go; Villanueva et al., 2002Go). Specifically, a BMP-dependent step precedes the migration of neural crest from the neural tube. The transcription factors Snail1 and Snail2 are known to regulate the EMT that is required for neural crest migration. We and others have found evidence that, CNS stem cells, which typically give rise to neuronal and glial fates (Johe et al., 1996Go), can be dorsally induced by BMPs to generate cells with neural crest characteristics (Alexanian and Sieber-Blum, 2003Go; Gajavelli et al., 2004Go; Mujtaba et al., 1998Go; Panchision et al., 2001Go; Rajan et al., 2003Go). In this study, we show that fetal telencephalic and adult CNS stem cells respond only to the combination of BMP2 and FGF2 by inducing Snail1 and Snail2, migrating from cortical explants, assuming a p75NGFR+ mesenchymal morphology and efficiently differentiating into smooth-muscle and non-CNS glia cells.

This finding is surprising for a number of reasons. First, key regional-identity regulators such as Foxg1 (Bf1), Nkx-2.1, Otx-1, Emx-2 and members of the Dlx gene family are expressed in the forebrain by E9.5 (Rubenstein et al., 1998Go), well before the ages we tested. Second, the neural crest is distinct from the CNS at cranial levels by approximately E9.5 (Serbedzija et al., 1992Go), at which point neural crest precursors have migrated out of the neuroepithelium and into the surrounding mesenchyme. Third, fate-mapping of chicken cells shows that neural crest does not arise from the telencephalic neuroepithelium during normal development, but instead arises from regions caudal to the epiphysis of the mid-diencephalon (Couly and Le Douarin, 1987Go; Le Douarin and Kalcheim, 1999Go).

However, the BMP-generated cells in our study have properties that are most similar to CPm, a cell type in close proximity to both CPe, a forebrain cell type, and cranial mesenchyme, a neural crest derivative. Both the migrating neural crest and CPm share expression of the Snail transcription factors (Marin and Nieto, 2004Go). Snail1 and Snail2 are centrally involved in cell movement, in particular during the epithelial to mesenchymal transition (EMT) of cells before migration (Barrallo-Gimeno and Nieto, 2005Go; De Craene et al., 2005Go). The expression of Snail genes in the neural crest of the posterior neural tube is transient; the last neural crest cells leave the neural tube at E9.5 in the mouse (Serbedzija et al., 1992Go), which corresponds in chicken development to the Hamburger and Hamilton (HH) stage 15 (Hamburger and Hamilton, 1992Go). However, studies in chicken show that expression of Snail and Slug in the CPm occur at HH stages 21-41 (Marin and Nieto, 2004Go), corresponding to approximately E12.5-18.5 in mouse gestation and beyond the time that neural crest emigrates from the neural tube. Chick-quail grafting experiments did not identify a contribution from either CPe (Wilting and Christ, 1989Go) or neural crest (Le Douarin and Kalcheim, 1999Go) to the CPm, leaving its developmental origins unclear. However, our results support the possibility that CPm might derive from anterior CNS cells.

Alternatively, grafting and genetic manipulation experiments suggest that CNS precursors can adopt new fates that are appropriate to their ectopic location in the CNS or PNS (Brustle et al., 1995Go; Hitoshi et al., 2002Go; Korade and Frank, 1996Go). Extensive dorsalization of cortex to Foxj1+ CPe, the most dorsal CNS cell type in the forebrain, occurs in transgenic mice expressing a constitutively active BMP receptor IA (c.a.BMPR-IA). Whereas CPe was the principal forebrain cell type generated in these animals, FGF2-expanded cortical stem cells transfected with the same c.a.BMPR-IA constructs very robustly generated p75NGFR+ precursors and smooth muscle (Panchision et al., 2001Go). Our current studies explain the difference between the transgenic and in-vitro data by showing a specific requirement for FGF2 signaling in the dorsalization of CNS stem cells to mesenchymal cell types. This suggests that the levels of FGF2 is limiting in this dorsal conversion in vivo. FGF signaling is crucial for supporting CNS development in vivo (Delaune et al., 2004Go; Rubenstein, 2000Go; Vaccarino et al., 1999Go). Since FGF2 is most frequently used as an obligatory mitogen during in-vitro stem cell expansion (Panchision and McKay, 2002Go), it is difficult to measure its interactions with other factors that regulate stem cell fate. Our explant culture assay shows that FGF2 is required for EMT and a neural-crest-like fate and that its actions are not duplicated by EGF or IGF1, even though these factors also promote proliferation.

FGF signaling has recently been implicated in the ventralization of neural stem cells and neural explants (Gabay et al., 2003Go; Kessaris et al., 2004Go; Kuschel et al., 2003Go). FGF2 expansion of cortical stem cells was shown to increase the frequency of Olig2+ cells, which mark ventral precursor cells that are capable of generating oligodendrocytes (Gabay et al., 2003Go). Whereas most oligodendrocyte generation is hedgehog-dependent (Kessaris et al., 2004Go; Tekki-Kessaris et al., 2001Go), an FGF2-dependent and hedgehog-independent pathway was also identified, suggesting that FGF2 acts in part by antagonizing BMP signaling (Chandran et al., 2003Go). Our results indicate that FGF2, rather than antagonizing dorsalization, is actually required for BMP2-mediated dorsalization. Thus, FGF2 does not appear to be ventralizing but rather acts as a permissive signal (Freeman and Gurdon, 2002Go) that allows stem cells to respond to other instructive cues. This idea is consistent with the requirement for FGF2 in such diverse responses like Wnt- and ß-catenin-mediated precursor proliferation (Israsena et al., 2004Go), hedgehog-mediated oligodendrocyte generation (Kessaris et al., 2004Go), induction of neural crest by paraxial mesoderm (Monsoro-Burq et al., 2003Go) and BMP-mediated dorsalization of CNS precursors to neural-crest-like cells (Gajavelli et al., 2004Go; Mujtaba et al., 1998Go; Panchision et al., 2001Go; Rajan et al., 2003Go).

We found that CNS stem cells from tissue of older embryos gave rise to increasing proportions of glia cells at the expense of smooth muscle and neurons. At E14.5, glial cells were generated only in regions of high local density in response to BMP treatment. By contrast, adult stem cells differentiated more uniformly to non-CNS glia. Previous studies show that these responses mirror the in-vivo developmental progression of cell-type differentiation (Panchision and McKay, 2002Go). Increased apoptosis followed by neuronal differentiation occurs in response to BMP receptor IB (BMPR-IB) activation in transgenic embryos (Panchision et al., 2001Go). Cultured neural precursors isolated from progressively older embryos also respond to BMPs with first apoptosis (Furuta et al., 1997Go; Graham et al., 1996Go), then neuronal differentiation in mid-gestation (Li et al., 1998Go; Mehler et al., 2000Go) and finally with glial differentiation in late-gestation and adulthood (Gross et al., 1996Go; Mabie et al., 1999Go). However, BMP treatment of EGF-expanded precursors generates glia cells with a CNS astrocyte-like morphology (Gross et al., 1996Go). We show that BMP treatment of FGF2-expanded precursors generates glia cells with non-CNS characteristics. An age-progressive bias in fate choice is also seen in PNS stem cells (Kruger et al., 2002Go; White et al., 2001Go). Our data suggest that the same mechanisms control the changing developmental bias for both CNS and non-CNS fates.

In conclusion, we show that both fetal and adult cortical stem cells have the capacity to generate neural-crest-like cells and that the age of the stem cells biases the type of derivatives that are generated. FGF2 is crucial for permitting this BMP-mediated response. Since many secreted molecules exert both morphogenic and mitogenic actions (Panchision and McKay, 2002Go), it will be of interest to determine whether these actions are mechanistically distinct. The ability of BMPs and FGFs to rapidly and efficiently re-direct the differentiation of CNS stem cells will allow further studies to define the mechanism of this interaction.


    Acknowledgments
 
We thank Story Landis, Vittorio Gallo, Tarik Haydar, Raji Padmanabhan, Raja Kittappa, Rea Ravin, Franziska Sailer and Andreas Androutsellis-Theotokis for advice and discussion, and Bechien Wu, Peter Warinner and Kimber Bogush for technical assistance. BMP ligands in initial experiments were provided by Wyeth Pharmaceuticals. We are grateful to Robert Adelstein (National Heart, Lung and Blood Institute, NIH, MD), Eric Turner (University of California, San Diego, CA) and Juan Archelos (University Hospital Graz, Austria, Europe) for their gifts of antibodies. M.H.M.S. was supported by a grant from the Swiss National Science Foundation (SNSF 3135-54876.98). This research was supported in part by the Intramural Research Program of the NIH, NINDS.


    Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/118/24/5849/DC1


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 Introduction
 Materials and Methods
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 BMP2-mediated activation of a...
 FGF2 and BMP2 differentiate...
 Both fetal and adult...
 Discussion
 References
 

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