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
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Summary |
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Key words: Forebrain, Neural stem cell, Cranial neural crest, Choroid plexus mesenchyme (CPm), Epithelial-mesenchymal transition (EMT), Snai1, Snai2
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Introduction |
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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, 1999b; Wilson and Rubenstein, 2000
). 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, 1999b
; Meulemans and Bronner-Fraser, 2004
). 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., 2002
; Panchision et al., 2001
). In the more posterior neural tube, BMPs induce roof plate and neural crest cells (Lee and Jessell, 1999a
). 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, 2002
; Knecht and Bronner-Fraser, 2002
; Trainor et al., 2002
; Wu et al., 2003
). 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, 2003
; Le Douarin and Kalcheim, 1999
; Meulemans and Bronner-Fraser, 2004
; Trainor et al., 2002
; Wu et al., 2003
).
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., 2004; Mujtaba et al., 1998
; Rajan et al., 2003
). This is in contrast to other studies that have shown CNS neuronal or glial differentiation after BMP treatment (Gross et al., 1996
; Li et al., 1998
; Mehler et al., 2000
), 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., 2004
) 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., 2005
; Gunhaga et al., 2003
). FGF2 and BMP2 treatment initially induces genes associated with the epithelial-mesenchymal transition (EMT) to a neural-crest-like state (Nieto, 2002
), 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).
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Materials and Methods |
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Culture of CNS stem cells
Both fetal and adult CNS stem cells were isolated and cultured as previously described (Johe et al., 1996; Kim et al., 2003
) in Dulbecco's modifies Eagle's medium (DMEM) F12 with N2 supplements (Kim et al., 2003
), 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., 2000
). 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., 2003). 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., 2001). 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
-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., 2003) 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.
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Results |
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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, 1996; Zorick et al., 1996
). 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., 1996; Vicario-Abejon et al., 2000
). 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., 2003
; Etchevers et al., 2002
; Marin and Nieto, 2004
; Nieto, 2002
). 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., 2002
). 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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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., 2005; Lee et al., 2004
). 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., 1991). 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., 2002), 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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BMP2-mediated activation of a Wnt signal and BMP2 expression |
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FGF2 and BMP2 differentiate CNS stem cells to multiple non-CNS derivatives |
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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., 2001), 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., 2003). 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., 2003
). 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, 1996
). 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, 2002
; Zorick et al., 1996
). This distinguishes them from oligodendrocytes of the intact adult brain that do not express p75NGFR under normal, non-injured circumstances (Beattie et al., 2002
). Myelin protein zero (MYP0), another gene expressed at high levels in mature Schwann cells (Jessen and Mirsky, 2002
), 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., 1999
). 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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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., 2002), 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., 1997
). 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, 1999
), 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
1 protein and Trp2 and collagen 2
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.
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Both fetal and adult CNS stem cells generate dorsalized derivatives |
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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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Discussion |
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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., 1998), 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., 1992
), 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, 1987
; Le Douarin and Kalcheim, 1999
).
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, 2004). 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, 2005
; De Craene et al., 2005
). 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., 1992
), which corresponds in chicken development to the Hamburger and Hamilton (HH) stage 15 (Hamburger and Hamilton, 1992
). However, studies in chicken show that expression of Snail and Slug in the CPm occur at HH stages 21-41 (Marin and Nieto, 2004
), 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, 1989
) or neural crest (Le Douarin and Kalcheim, 1999
) 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., 1995; Hitoshi et al., 2002
; Korade and Frank, 1996
). 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., 2001
). 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., 2004
; Rubenstein, 2000
; Vaccarino et al., 1999
). Since FGF2 is most frequently used as an obligatory mitogen during in-vitro stem cell expansion (Panchision and McKay, 2002
), 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., 2003; Kessaris et al., 2004
; Kuschel et al., 2003
). 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., 2003
). Whereas most oligodendrocyte generation is hedgehog-dependent (Kessaris et al., 2004
; Tekki-Kessaris et al., 2001
), an FGF2-dependent and hedgehog-independent pathway was also identified, suggesting that FGF2 acts in part by antagonizing BMP signaling (Chandran et al., 2003
). 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, 2002
) 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., 2004
), hedgehog-mediated oligodendrocyte generation (Kessaris et al., 2004
), induction of neural crest by paraxial mesoderm (Monsoro-Burq et al., 2003
) and BMP-mediated dorsalization of CNS precursors to neural-crest-like cells (Gajavelli et al., 2004
; Mujtaba et al., 1998
; Panchision et al., 2001
; Rajan et al., 2003
).
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, 2002). Increased apoptosis followed by neuronal differentiation occurs in response to BMP receptor IB (BMPR-IB) activation in transgenic embryos (Panchision et al., 2001
). Cultured neural precursors isolated from progressively older embryos also respond to BMPs with first apoptosis (Furuta et al., 1997
; Graham et al., 1996
), then neuronal differentiation in mid-gestation (Li et al., 1998
; Mehler et al., 2000
) and finally with glial differentiation in late-gestation and adulthood (Gross et al., 1996
; Mabie et al., 1999
). However, BMP treatment of EGF-expanded precursors generates glia cells with a CNS astrocyte-like morphology (Gross et al., 1996
). 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., 2002
; White et al., 2001
). 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, 2002), 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.
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Acknowledgments |
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Footnotes |
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References |
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