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First published online March 13, 2008
Stem Cells Vol. 26 No. 6 June 2008, pp. 1565 -1574
doi:10.1634/stemcells.2007-0832; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Fibroblast Growth Factor 2 Maintains the Neurogenic Capacity of Embryonic Neural Progenitor Cells In Vitro but Changes Their Neuronal Subtype Specification

Angela Bithell, Sophie E. Finch, Matthew F. Hornby, Brenda P. Williams

Centre for the Cellular Basis of Behaviour, Medical Research Council Centre for Neurodegeneration Research, James Black Centre, Institute of Psychiatry, King's College London, London, United Kingdom

Key Words. Transcription factor • Neural differentiation • Fibroblast growth factor 2 • Lineage specification • Growth factors

Correspondence: Correspondence: Brenda P. Williams, Ph.D., Centre for the Cellular Basis of Behaviour, MRC Centre for Neurodegeneration Research, The James Black Centre, King's College London, Institute of Psychiatry, 125 Coldharbour Lane, London SE5 9NU, United Kingdom. Telephone: 44-(0)207-848-0097; Fax: 44-(0)207-848-5308; e-mail: b.williams{at}iop.kcl.ac.uk

Received on October 1, 2007; accepted for publication on February 29, 2008.

First published online in STEM CELLS EXPRESS  March 13, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Many in vitro systems used to examine multipotential neural progenitor cells (NPCs) rely on mitogens including fibroblast growth factor 2 (FGF2) for their continued expansion. However, FGF2 has also been shown to alter the expression of transcription factors (TFs) that determine cell fate. Here, we report that NPCs from the embryonic telencephalon grown without FGF2 retain many of their in vivo characteristics, making them a good model for investigating molecular mechanisms involved in cell fate specification and differentiation. However, exposure of cortical NPCs to FGF2 results in a profound change in the types of neurons generated, switching them from a glutamatergic to a GABAergic phenotype. This change closely correlates with the dramatic upregulation of TFs more characteristic of ventral telencephalic NPCs. In addition, exposure of cortical NPCs to FGF2 maintains their neurogenic potential in vitro, and NPCs spontaneously undergo differentiation following FGF2 withdrawal. These results highlight the importance of TFs in determining the types of neurons generated by NPCs in vitro. In addition, they show that FGF2, as well as acting as a mitogen, changes the developmental capabilities of NPCs. These findings have implications for the cell fate specification of in vitro-expanded NPCs and their ability to generate specific cell types for therapeutic applications.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Dorsoventral (D-V) patterning of the telencephalon is induced by gradients of diffusible signaling molecules secreted from the surrounding tissues, such as sonic hedgehog (Shh), fibroblast growth factor 8 (FGF8), WNTs, and bone morphogenic proteins (BMPs) [1–5]. The concentration gradients established by these signaling molecules are responsible for initiating the expression of specific sets of transcription factors in populations of neural progenitor cells (NPCs) along the D-V axis [6]. NPCs within each domain generate specific types of neurons and glia in a precise temporal sequence, and many of these cells migrate via complex routes to their final destination (reviewed in [7–10]).

NPCs of the embryonic cortex specifically express transcription factors able to confer a dorsal cell fate; these include Pax6, Emx2, and Ngn2 [11–21]. For example, Pax6 and Emx2 are indispensable for the specification of cortical neuroblasts and the generation of glutamatergic neurons [21]. Conversely, ventral-specific transcription factors, including Dlx1, Dlx2, Dlx5, Gsh2, Mash1, Lhx6, and Nkx2.1 [12, 18, 22–29], are responsible for the specification and differentiation of subpallial NPCs into cholinergic neurons [25], together with a diverse range of GABAergic neurons [30] that migrate to populate both subpallial and pallial domains, as well as the olfactory bulb and hippocampus [31–36]. Some oligodendrocyte progenitor cells (OPCs) generated in the ventral telencephalon also migrate dorsally into the cortex [35, 37, 38]. This long-distance migration of large numbers of cortical GABAergic neurons and OPCs is necessary because the correct development of these cell types requires signals from the ventral telencephalon and the subsequent expression of ventral-specific transcription factors [37, 39–43]. However, we still do not fully understand the mechanisms by which expression of specific combinations of transcription factors directs NPCs to produce specific neural cell types.

In recent years, advances in neural stem cell (NSC) research have raised hope for cell replacement therapies. However, for these therapies to be successful, transplanted cells must be able to differentiate into particular types of neurons or glia. For this to occur, it is possible that the molecular mechanisms important for the generation of different neural cells during embryonic development must be recapitulated. Since cells need to be expanded in vitro before transplantation, the effects of growth factors, such as FGF2 (a mitogen and known survival factor), on the molecular and cellular phenotype of NPCs may have important implications for their ability to respond to local cues and their subsequent fate.

Here, we use retroviral lineage tracing to show that NPCs isolated from different regions of the developing telencephalon display many characteristics of their in vivo counterparts, both in their molecular phenotypes and in their differentiated fates. This makes them a useful tool to examine the complex mechanisms involved in generating neural diversity in the forebrain. However, exposure of cortical NPCs to FGF2 results in a molecular phenotype more appropriate to NPCs isolated from the ganglionic eminences (GE NPCs) [44–47]. These cells now undergo spontaneous neurogenesis upon FGF2 withdrawal, generating significant numbers of GABAergic rather than glutamatergic neurons. These observations have important implications for the generation of NPC populations designed for cell-replacement strategies, since in vitro conditions may affect their competence to generate particular types of neural cells required therapeutically.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals and Cell Culture
The cerebral cortices or ganglionic eminences (both lateral ganglionic eminences [LGE] and medial ganglionic eminences [MGE]) of embryonic day (E) 14 or 17 Sprague-Dawley rats were dissected in cold Hanks' balanced saline solution (Gibco, Grand Island, NY, http://www.invitrogen.com) and processed as described previously [48, 49]. Briefly, E14 tissue was incubated in trypsin/EDTA (Gibco) plus 0.001% DNase I (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 10 minutes at 37°C, and the reaction was stopped by addition of 10% fetal calf serum or trypsin inhibitor (Sigma-Aldrich) before dissociation into a single-cell suspension. Cells grown in 24-well plates were plated onto poly-D-lysine (Sigma-Aldrich)-coated, 13-mm-diameter glass coverslips.

Clonal Analysis
NPC cultures were infected with the BAG retrovirus, containing the lacZ gene encoding β-galactosidase [50], by the addition of virus directly to the culture medium such that single infected NPCs and their progeny could be identified by expression of β-galactosidase [48, 49, 51]. Culture medium was replaced the following day, and the following appropriate factors were added to experimental wells: platelet-derived growth factor BB (PDGFBB; 30 ng/ml; Insight Biotechnology), cilliary neurotrophic factor (CNTF; 30 ng/ml; Insight Biotechnology, London, http://www.insightbio.com), and FGF2 (10 ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com). After an additional 24 hours, factors were removed, and the cells were maintained for an additional 4 days in defined medium to differentiate, unless otherwise stated. Throughout the culture period, half of the medium was replaced every 2–3 days. Cultures were fixed in 4% paraformaldehyde, and the number and types of clones were analyzed by triple fluorescence immunocytochemistry. For GABA and glutamate immunostaining, 0.1% glutaraldehyde was added to the fixation step.

Fluorescence Immunocytochemistry

Analysis of Starting Cell Population.   The composition of the starting cell population at E14 and E17 was determined on the day following plating using antibodies that recognize nestin (NPCs), βIII-tubulin (neurons), O4 (oligodendrocytes), and glial fibrillary acidic protein (astrocytes), as described below.

5-Bromo-2'-Deoxyuridine Assay.   Cells were given a 4-hour pulse of 5-bromo-2'-deoxyuridine (BrdU) following 1 day in vitro (1DIV), identified using anti-BrdU antibodies, and colabeled with Ki67 and 4,6-diamidino-2-phenylindole, as detailed in the supplemental online Materials and Methods.

Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling.   Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI, http://www.promega.com), according to the manufacturer's instructions (also described in the supplemental online Materials and Methods).

Transcription Factor Expression and Clonal Analysis.   Immunocytochemistry was performed as described previously [48, 49, 51]. Details of primary and secondary antibodies are given in the supplemental online Materials and Methods. Cells were mounted in Prolong antifade mounting medium (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) and analyzed using a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, http://www.nikon.com) with Lucia G Imaging software (Laboratory Imaging Ltd., Prague, CZ, http://www.lim.cz/en) or a Zeiss AxioImager Z1 with Axiovision 6.3 imaging software (Carl Zeiss, Jena, Germany, http://www.zeiss.com) and Adobe Photoshop (Adobe Systems Inc., San Jose, CA, http://www.adobe.com). Statistical analysis was performed using SPSS (SPSS, Chicago, http://www.spss.com) and Excel (Microsoft, Redmond, WA, http://www.microsoft.com).

Anti-Phospho-ERK Immunocytochemistry.   Following exposure of cultures to PDGFBB for 15 minutes, activated mitogen-activated protein kinase ([MAPK]: phosphorylated-extracellular signal regulated kinases 1 and 2, [phospho-ERK1 and ERK2]) was detected using rabbit polyclonal IgG dual-phosphorylated anti-active MAPK antibody (Promega) exactly according to the manufacturer's instructions.

Western Blot Analysis
Following 15 minutes of exposure to PDGFBB or CNTF, cultures were rinsed in cold Tris-buffered saline containing phosphatase inhibitors before preparation of protein lysates. Samples were run on 7.5% SDS-polyacrylamide gel electrophoresis gels and blotted onto polyvinylidene difluoride membrane (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Membranes were blocked in 1% skim milk before incubation with the following primary antibodies: anti-active MAPK (1:10,000; rabbit polyclonal IgG; Promega), anti-phosphorylated-signal transducers and activators of transcription 3 ([anti-p-STAT3]; 1:200, rabbit polyclonal IgG; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and anti-glyceraldehyde-3-phosphate dehydrogenase ([anti-GAPDH]; 1:3,000, rabbit polyclonal IgG; Abcam, Cambridge, MA, http://www.abcam.com). Primary antibodies were visualized using anti-rabbit-IgG conjugated to horseradish peroxidase (1:5,000; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) followed by incubation in Lumi-light substrate (Roche Diagnostics) and detected by exposure to Lumi-Light chemiluminescence film (Roche Diagnostics).

RNA Extraction and Polymerase Chain Reaction Analysis
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) with a DNase I step. Two micrograms of total RNA was used to synthesize first strand cDNA using oligo(dT)15 (Promega) and PowerScript reverse transcriptase (Clontech, Palo Alto, CA, http://www.clontech.com). Standard polymerase chain reaction (PCR) was performed using Taq polymerase and the primers given in the supplemental online Materials and Methods.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Nonexpanded NPCs In Vitro Generate Neurons Appropriate to Their In Vivo Region of Origin
E14 cortical NPCs require extracellular signals to differentiate when grown in defined medium in vitro; exposure to PDGFBB induces neuronal differentiation, whereas CNTF induces astrogliogenesis [48, 49]. To determine whether NPCs isolated from the ventral telencephalon also generate neurons under similar conditions, we performed a clonal analysis on E14 GE NPCs and compared the number of neurons generated in the presence or absence of PDGFBB. In addition, we determined whether NPCs isolated from these two different forebrain regions retain the positional identity imparted in vivo such that cortical NPCs give rise to glutamatergic neurons and GE NPCs give rise to GABAergic neurons [52, 53].

The percentages of glutamatergic or GABAergic neuronal clones generated in the presence or absence of PDGFBB are shown in Figure 1A, and examples of each clone type are shown in Figure 1B–1E. In the cortex, the percentage of GABAergic neuronal clones did not increase significantly with the addition of PDGFBB, whereas the percentage of glutamate-positive clones increased from 43% to 68% (p < .001). In sharp contrast, GE NPCs generated few if any glutamatergic neuronal clones when exposed to PDGFBB, whereas the percentage of GABAergic neuronal clones significantly increased, from 51% to 81% (p < .01). Thus, although PDGFBB instructs both dorsal and ventral NPCs to undergo neurogenesis, the type of neurons generated reflects the in vivo origin of the NPCs along the D-V axis.


Figure 1
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  		Figure 1. Embryonic day 14 neural progenitor cells differentiate into appropriate neuronal subtypes. (A): The number of GABA or Glut clones is shown as a percentage of total neuronal clones. (B–E): Top panels show a single-cell β-Gal-positive clone ([B], white arrow) that is GABA ([C], white arrow). Bottom panels show a single-cell β-Gal-positive clone ([D], white arrow) that is Glut ([E], white arrow). Abbreviations: GABA, GABAergic; β-Gal, β-galactosidase; GE, ganglionic eminence; Glut, glutamatergic; PDGFBB, platelet-derived growth factor BB.

 
E17 Cortical NPCs Generate Glia in Response to Exogenous PDGFBB
By E17 in vivo, cortical neurogenesis is slowing down, and there is a switch toward gliogenesis that continues into postnatal stages [54]. We therefore investigated the neurogenic capacity of cortical NPCs from E17 compared with those from E14 in response to PDGFBB to determine whether the two populations exhibited intrinsic differences in their developmental potential.

NPCs were isolated from E14 or E17 cortices and a retroviral clonal analysis performed (Table 1; supplemental online Fig. 1). Whereas E14 cortical NPCs underwent neuronal differentiation in response to PDGFBB, with the percentage of neuronal clones increasing from 28% to 79% (p < .01), E17 NPCs did not. However, E17 NPCs showed a small but significant increase in oligodendrocyte clones (from 9% to 15%; p < .05) that may reflect the ability of PDGFBB to activate PDGF alpha receptors (PDGF{alpha}Rs) expressed by OPCs [55] present in the cortex at E17 following migration from the ventral telencephalon during development [35, 56]. In all cases, we observed a decrease in NPC clones in the presence of PDGFBB (p < .05), concomitant with an increase in clones composed of differentiated cell types.


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  		Table 1. NPC response to PDGFBB changes with time

 
This loss of a neurogenic response is unlikely to be due to the loss of PDGF beta receptor (PDGFβR) expression by E17 cortical NPCs, since immunofluorescence labeling showed that the vast majority (87%) of cells express PDGFβR (supplemental online Fig. 2), compared with essentially 100% at E14, as we have previously reported [48] (supplemental online Fig. 2). Moreover, a short (15-minute) exposure of E17 cortical NPCs to PDGFBB resulted in a robust activation of downstream MAPK signaling (Fig. 2), shown by the appearance of pERK1- and pERK2-immunopositive cells (Fig. 2A) and by Western blot of cell lysates (Fig. 2B). Furthermore, E17 cortical NPCs were capable of differentiating in response to other exogenous signals in vitro, generating astrocytes in response to CNTF via STAT3 signaling as E14 cortical NPCs do (supplemental online Table 1 and supplemental online Fig. 3, respectively) [49, 57]. Therefore, at E17, despite the presence of active PDGFβRs, cortical NPCs no longer respond to PDGFBB by generating neurons but instead show a small increase in glial differentiation, recapitulating the switch in vivo at this stage from neurogenesis to gliogenesis.


Figure 2
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  		Figure 2. Activation of MAPK signaling in neural progenitor cells (NPCs) following PDGFBB exposure. (A): NPCs from E17 or E14 telencephalon showed a dramatic increase in active (dual-phosphorylated) MAPK following exposure to PDGFBB for 15 minutes. (B): Western blot analysis from sister cultures showed increased phospho-ERK1 and ERK2 (MAPK) following exposure of E14 or E17 cortical NPCs to PDGFBB. Abbreviations: E, embryonic day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDGF BB, platelet-derived growth factor BB.

 
E14 + 3DIV Cortical NPCs No Longer Undergo Neurogenesis upon Exposure to PDGFBB
The capacity of NPCs to generate neurons is known to decrease as embryonic development proceeds, whereas gliogenesis increases (reviewed in [54]). Our above results suggest, at least in part, that this restriction in developmental potential is retained when NPCs isolated from different developmental ages are cultured in our in vitro system. We therefore investigated whether such a change occured in vitro by maintaining E14 cortical NPCs in defined medium for 3 days (E14 + 3DIV) to generate E17-equivalent NPCs before determining their response to PDGFBB or CNTF using retroviral lineage analysis.

We observed no significant increase in the percentage of neuronal or glial clones following E14 + 3DIV NPC exposure to PDGFBB compared with controls. However, as with E17 NPCs, E14 + 3DIV NPCs retained their ability to respond to CNTF (Table 2), with a significant increase in astrocyte clones (from 3% to 23%; p < .05) and mixed astrocyte/oligodendrocyte clones detected compared with controls (from 0% to 8%; p < .05). These results suggest that cell fate changes that take place between E14 and E17 in vivo can be mimicked in vitro. However, E14 + 3DIV NPCs lack the PDGFBB-mediated gliogenic response shown by E17 NPCs.


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  		Table 2. The change in NPC response to PDGFBB with developmental age also occurs in vitro

 
The Transcription Factor Profile of NPCs Changes with Both DIV and Exposure to FGF2
Since E14 cortical NPCs in vitro generate neuronal subtypes appropriate for their region of origin and show time-dependent changes in their response to extracellular signaling, we next determined whether these findings correlated with their expression of pivotal, dorsal-specific transcription factors (TFs) that promote a glutamatergic neuronal fate in vivo and whether TF expression changed with DIV. We further investigated whether exogenous FGF2 influenced the TF expression and subsequent cell fate determination of NPCs in our culture system, since NSCs maintained in FGF2 retain their ability to generate neurons, although their regional specification may be altered [44–46, 58–60].

E14 cortical NPCs grown in the presence (+FGF2) or in the absence (–FGF2) of 10 ng/ml FGF2 were analyzed following 1DIV and 3DIV by PCR and immunocytochemistry to determine mRNA and protein expression of a number of region-specific TFs (Figs. 3 and 4, respectively) and also mRNA expression of Shh, Bmp4, and noggin (Fig. 3). GE NPCs grown under identical conditions were used for comparison. E14 cortical NPCs + 1DIV –FGF2 expressed dorsal-specific TFs, including Emx1, Emx2, and Pax6, but no detectable levels of most ventral-specific TFs analyzed, although they did express low levels of Mash1 (Fig. 3). Exposure to FGF2 for 1DIV did not appreciably change transcript levels of the genes analyzed. In comparison, E14 GE NPCs showed high expression levels of most ventral-specific TFs investigated (Fig. 3). Although Pax6 expression was also detected in GE cultures, less than 6% of cells expressed protein (Fig. 4), and some of these cells were neurons, not NPCs (A.B. and S.E.F., unpublished observations). At 3DIV, despite a relatively unchanged gene expression profile in cortical NPCs –FGF2, those +FGF2 showed readily detectable levels of many ventral-specific TFs, including Dlx2, Mash1, and Olig2, while still expressing Emx1, Emx2, and Pax6. Comparison of the TF expression profile of cortical NPCs +FGF2 at 3DIV with GE NPCs –FGF2 at 3DIV showed remarkable similarities, with the exception of Lhx6 and Emx1, suggesting that FGF2 reprograms positional specification. Furthermore, we observed that Bmp4 expression was increased in both cortical and GE NPCs at 3DIV +FGF2 and that low levels of noggin were detected in GE NPCs +FGF2.


Figure 3
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  		Figure 3. mRNA expression of region-specific transcription factors and signaling proteins in neural progenitor cells (NPCs). Polymerase chain reaction analysis from embryonic day 14 Ctx or GE NPCs after 1DIV or 3DIV in the presence (+) or absence (–) of fibroblast growth factor 2 (FGF2) revealed significant upregulation of ventral-specific transcription factors in Ctx NPCs +FGF2. Amplification cycles were as follows: Gapdh and TFs, 30 cycles (except for Dlx5, 40 cycles); all other primer sets, 40 cycles. Results represent consistent findings from independent biological samples. Black and gray text shading indicates samples analyzed in the same experiment. Abbreviations: Ctx, cortical; DIV, days in vitro; GE, ganglionic eminence.

 


Figure 4
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  		Figure 4. FGF2 leads to ventralization of cortical neural progenitor cells (NPCs) in vitro. Embryonic day 14 cortical (A) or GE NPCs (B) were analyzed for expression of transcription factors following 1DIV and 3DIV +FGF2 or –FGF2. Results are shown as percentages of 4,6-diamidino-2-phenylindole-positive cells and are from at least three independent experiments. Exposure of cortical NPCs to FGF2 resulted in significant increases in the percentage of cells expressing MASH1 and OLIG2. Error bars show SDs. Significant changes are indicated: *, p < .01; **, p < .001. For clarity, significant changes from 1DIV and 3DIV in (A) are not depicted but were as follows: OLIG2 +FGF2, p < .001; PAX6 –FGF2, p < .001; NGN2 both +FGF2 and –FGF2, p < .01. Abbreviations: DIV, days in vitro; FGF, fibroblast growth factor; GE, ganglionic eminence.

 
We determined expression of the dorsal-specific TFs NGN2 and PAX6 and the ventral-specific TFs MASH1 and OLIG2 at the protein level (Fig. 4). Following 1DIV, there was no significant difference in the percentage of PAX6-positive cells in any condition. However, after 3DIV, the percentage of PAX6-positive cells in cortical cultures –FGF2 was significantly lower than in those +FGF2 (28% and 56%, respectively; p < .00). Following 1DIV, the percentage of NGN2-expressing cells was significantly reduced +FGF2 (4% compared with 20% –FGF2; p < .00), although percentages were comparable following 3DIV. Strikingly, the percentage of cortical cells expressing the ventral-specific TF MASH1 rose more than threefold following 1DIV +FGF2 compared with –FGF2 (p < .00), with no significant change observed in GE NPCs. A significant increase in OLIG2-expressing cells was also observed after 1DIV +FGF2 in both cortical and GE NPCs (p < .00 for both). This increase was greater in cortical NPCs after 3DIV, with almost 20% OLIG2-positive cells compared with less than 7% at 1DIV (p < .00). Some changes in TF expression also occurred with increasing DIV in the absence of FGF2; most notably, there was a significant decrease in PAX6-positive cells in cortical cultures from 54% at 1DIV to 30% after 3DIV (p < .01), and NGN2-positive cells decreased from 18% following 1DIV to 10% at 3DIV (p < .01). Interestingly, E17 cortical NPCs analyzed after 1DIV showed proportions of TF-expressing cells similar to those of E14 + 3DIV (supplemental online Fig. 4).

We also observed the percentage of cells expressing particular TF combinations to determine whether dorsal and ventral TFs were coexpressed. Most MASH1-positive cortical NPCs coexpressed PAX6 in all conditions. Although cells coexpressing MASH1/OLIG2 were rarely seen in cortical cultures –FGF2, some cells exhibited this phenotype after 1DIV +FGF2 and constituted more than 20% after 3DIV. Few cells coexpressed OLIG2/NGN2 in any condition (data not shown).

Thus, with increasing DIV, cortical NPCs in the absence of FGF2 downregulated TFs important for the generation of glutamatergic neurons, providing a possible explanation for the loss of neuronal differentiation in E14 + 3DIV NPCs. Exposure to FGF2 led to an increase in NPCs expressing ventral-specific TFs but appeared to maintain high percentages of PAX6-positive cells, leading to a significant number of NPCs coexpressing dorsal- and ventral-specific TF combinations.

Exposure of E14 + 3DIV Cortical NPCs to FGF2 Leads to PDGFBB-Independent Generation of GABAergic Neurons
To evaluate the effect of changes in TF expression on NPC fate, we infected E14 cortical NPCs with the BAG retrovirus on the day of plating and grew them in the presence or absence of 10 ng/ml FGF2 for 3DIV. At this stage, some cells were fixed, and the resultant clones were analyzed for TF expression by immunofluorescence (Fig. 5A, 5B). The remaining cell cultures were switched into defined medium in the presence or absence of 30 ng/ml PDGFBB to induce neuronal differentiation. Following an additional 5DIV, cells were fixed, and clonal analysis was performed to determine the neuronal subtypes (glutamatergic or GABAergic) generated by NPCs (Fig. 5A, 5C).


Figure 5
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  		Figure 5. Cortical neural progenitor cells (NPCs) undergo PDGFBB-independent GABA neuronal differentiation following FGF2 signaling. Embryonic day (E) 14 cortical NPCs were infected with the BAG retrovirus on the day of plating (D0) and grown for an additional 3DIV in the presence or absence of 10 ng/ml FGF2. Some were then cultured for an additional 5DIV with or without PDGFBB for the first 24 hours (A). E14 cortical NPC clones were analyzed for expression of TFs at 3DIV (± FGF2) (A, B) and neuronal subtype markers at 3DIV/5DIV (+/–FGF2/+/–PDGFBB) (A, C). Examples of clones from (B, C) are shown in (D). A minimum of 150 clones per condition were counted at 3DIV (B) and 80 clones at 3DIV/5DIV (C). The percentages of clones expressing a particular TF are listed at the top of (B). Pie-charts in (B) show percentages of clones containing combinations of TF expression. (Letter combinations [e.g., MO], represent double-labeled cells; letters separated by a slash[/] represent both cell types present in the clone, such as M/MO [MASH1 only and MASH1/OLIG2 double-positive]). In the bottom histogram in (C), clones were classed as GABA (GABA+) or non-GABA (GABA–). In the top histogram, clones were classed as glutamatergic (vGlut1/2+) or nonglutamatergic (vGlut1/2–). The top four panels in (D) show an eight-cell clone that is OLIG2-positive (NGN2-negative), and the bottom three panels show a single-cell, GABA neuronal clone. Scale bars= 25 µm. Abbreviations: β-gal, β-galactosidase; DIV, days in vitro; FGF, fibroblast growth factor; GABA, GABAergic; M, MASH1; N, NGN2; O, OLIG2; P, PAX6; PDGFBB, platelet-derived growth factor BB; TF, transcription factor.

 
Following 3DIV, as in whole population analyses (Fig. 4), addition of FGF2 led to upregulated expression of MASH1 and OLIG2 in a large percentage of β-galactosidase-positive clones (Fig. 5B, top of each column shows the percentages of all clones assessed for a particular TF that contained positive cells): clones containing MASH1-positive cells increased to 51.8% from 6.1%, clones containing OLIG2-positive cells increased to 61.8% from 0%, and clones containing PAX6-positive cells increased to 67.4% from 32.8%. In addition to ranking a clone as positive or negative for a single TF, many of the immunostaining combinations revealed heterogeneity within a clone (Fig. 5B, pie charts). Such combinations may be important in determining different fates of cells within the clone. For example, MASH1-positive clones +FGF2 accounted for more than 50% of total clones, but many of the MASH1-positive cells coexpressed OLIG2 or PAX6, and some clones were composed of cells expressing each TF alone and also in combination (Fig. 5B).

In agreement with our earlier findings that E14 + 3DIV cortical NPCs do not generate neurons in response to PDGFBB (Table 2), there was no significant increase in clones containing glutamatergic cells (vGlut1/2-positive) or GABAergic cells when cultures were grown in the absence of FGF2 (Fig. 5C, top and bottom panels, respectively). In striking contrast, we observed a significant increase in GABA-containing clones, from 7% to 54.2% (p < .05) (Fig. 5C), following 3DIV +FGF2. An example of a GABAergic clone is shown in Figure 5D. No significant difference in vGlut1/2 clones was observed.

To ensure that the NPC fate changes observed in the presence of FGF2 were not due to the known survival actions of this mitogen [61], we assessed proliferation and cell death in our system. At 1DIV +/–FGF2, a 4-hour pulse of BrdU was given to E14 cortical cultures, which were subsequently fixed and analyzed for expression of BrdU and Ki67. FGF2 significantly increased the labeling index from 58.3 to 69.5 (supplemental online Fig. 5). At 3DIV, analysis of clones using TUNEL showed no significant decrease in cell death within clones +FGF2 compared with –FGF2 (supplemental online Fig. 5), but the average clone size increased (7.25 ± 0.2 cells +FGF2 compared with 3.5 ± 0 cells –FGF2), most likely explained by the increased rate of proliferation, and there were on average 8.3% ± 1.3% more clones +FGF2 than –FGF2. The latter finding we attribute to increased retroviral infection due to the shorter cell-cycle length +FGF2. Following an additional 5DIV +/–PDGFBB, there was still no significant difference in cell death within clones among all four conditions using either TUNEL or active (cleaved) caspase-3 labeling (supplemental online Fig. 5). Dying cells generally represented only a small percentage of a positive clone. FGF2 has also previously been shown to promote NPC differentiation into glial cells [62]. We therefore also determined the percentage of clones that contained oligodendrocytes, astrocytes, and neurons, as well as mixed clones (supplemental online Fig. 6). We found a significant increase in clones containing glia, particularly astrocytes, following 3DIV +FGF2, and this increase was not enhanced by the further addition of PDGFBB. In agreement with our previous data (Table 2), the increase in glial clones with PDGFBB was variable and not significant.

Thus, following 3DIV +FGF2, cortical NPCs can differentiate into neurons, but their phenotype is switched from glutamatergic to GABAergic (Fig. 5), and an increase in gliogenesis is observed (supplemental online Fig. 6). Interestingly, we noted that a large number of single-cell clones in both conditions with FGF2 were GABAergic, compared with relatively few with PDGFBB alone and none in the absence of both factors (data not shown), possibly suggesting reprogramming of neuroblasts. Surprisingly, there was a highly significant increase in the percentage of GABA clones following 3DIV +FGF2 in the absence of PDGFBB (71.2% in +FGF2/–PDGFBB, compared with 1.1% in –FGF2/–PDGFBB cultures; p= .001; Fig. 5) and in glial-containing clones (supplemental online Fig. 6), suggesting that these NPCs no longer need an additional signal to differentiate into neurons or glia but do so spontaneously following removal of FGF2.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosure of Potential...
 Acknowledgments
 References
 
Here we describe the use of clonal analysis to investigate the relationship between the expression of TFs that specify the fate of embryonic NPCs in vivo and their competence to generate particular types of neurons and glial cells in vitro. We report that nonexpanded telencephalic NPCs isolated at the peak of neurogenesis (E14) express appropriate region-specific TFs and readily differentiate into neurons appropriate for their region of origin following exposure to PDGFBB. However, this neurogenic capacity is lost over time and correlates with a decrease in expression of PAX6 and NGN2. In contrast, exogenous FGF2 leads to reprogramming of E14 cortical NPCs and significant GABAergic neuronal differentiation that is independent of PDGFBB signaling.

Nonexpanded NPCs In Vitro Show Time-Dependent Changes in Response to Extracellular Signals
From E12 to E18 in the rat telencephalon, a diverse range of neuronal cell types is generated from distinct regions of the germinal zones along the dorsoventral axis (reviewed in [7]). Around E17, NPCs switch from generating predominantly neurons to producing glia [54, 62, 63, 64], and this switch is governed by TFs [37, 65–67], growth factor receptors, and extracellular growth factors [64, 68, 69].

We, and others, have previously shown that NPCs grown in culture require an extracellular signal to differentiate; NPCs cells isolated from E14 rat cerebral cortex and grown in dissociated cell culture generate neurons when exposed to PDGFBB and astrocytes when exposed to CNTF [48, 49, 57, 70]. Here we show that E17 cortical NPCs no longer generate neurons in response to PDGFBB and instead begin to produce glia. We suggest that this change corresponds to the decline in neurogenesis and onset of gliogenesis observed around E17 in vivo [68]. Interestingly, clonal analysis of E14 + 3DIV cortical NPCs reveals that NPCs lose their ability to generate neurons in response to PDGFBB in vitro, coincident with a decrease in Pax6 expression and Ngn2 expression, which are key determinants of cortical NPC fate [17, 18, 21, 59].

NPCs residing in different dorsoventral locations along the neuroaxis also give rise to different neural cell types, including subtypes of neurons. In the dorsal telencephalon, cortical NPCs generate cortical glutamatergic neurons, whereas cortical GABAergic neurons are born in the MGE and LGE and migrate into the cortex [26, 29–33]. Previously, we reported that E14 cortical NPCs generate neurons in response to PDGFBB [48, 49]. We now show that NPCs from the GE do likewise but that cortical NPCs predominantly generate glutamatergic neurons, whereas GE NPCs almost exclusively differentiate into GABAergic neurons.

Thus in vitro, dissociated, nonexpanded NPCs display characteristics of NPCs in vivo in both their spatial and temporal fate specification, producing cell types appropriate to their region of origin and time of isolation and reflected by their expression of region-specific TFs. As such, they provide a useful in vitro tool with which to investigate the molecular mechanisms of neuronal subtype specification.

FGF2 Reprograms the Positional Specification Profile of E14 NPCs
Combinatorial expression of important transcription factors imparts positional identity in vivo, facilitating the generation of specific neurons and glia in different brain regions [29–30, 71–73]. The potential of NPCs for specific cell-replacement strategies relies on their ability to differentiate into particular neural phenotypes in a given transplantation paradigm [74]. Since it is a prerequisite that NPCs for transplantation can be readily expanded in vitro, it is imperative that we fully understand the implications of the expansion process on NPC fate. This includes understanding the effects of the mitogen FGF2 on NPC respecification, particularly in light of conflicting evidence regarding the extent to which it can do so [45, 46, 60, 75, 76].

Comparing the in vitro gene expression profile of cortical NPCs in the presence or absence of FGF2, our findings agree with those of others that, in addition to acting as a mitogen (supplemental online Fig. 6), FGF2 is capable of changing the molecular phenotype of NPCs, leading to a more ventral telencephalic expression profile [46]. We extended these observations to show that the changes in TF expression closely correlate with subsequent NPC fate, directing the NPCs almost exclusively along a GABAergic rather than glutamatergic pathway. These finding are not attributable to a selective survival effect of FGF2 for GABAergic neuronal clones in our cortical NPC cultures (supplemental online Fig. 5), and GABAergic neuronal differentiation of GE NPCs occurs readily in the absence of FGF2 under identical conditions (Fig. 1). This phenotypic change is most likely due to loss of expression of TFs, including Ngn2, and upregulation of the proneural TF, Mash1. In vivo, Ngn2 and Pax6 are important for glutamatergic cell fate specification in the cortex and for repressing ventral cell fate; their loss results in ectopic expression of ventral-specific transcription factors and the generation of GABAergic neurons [14, 16, 18, 77, 78]. Conversely, in the ventral telencephalon, several ventral-specific transcription factors, including Mash1, play crucial roles in the specification and differentiation of GABAergic interneurons [14, 23, 29, 79, 80]. Our results show that in the absence of FGF2, E14 cortical NPCs retain their positional identity and hence generate glutamatergic neurons. We report that FGF2 has a dramatic ventralizing effect on E14 cortical NPCs, and after as little as 3DIV, their gene expression profile is remarkably similar to that of GE NPCs. However, they retain some dorsal TF expression, particularly Pax6, which argues for deregulation of positional identity rather than simple ventralization, and we observe that many cells coexpress dorsal and ventral TFs. However, it is noteworthy that not all NPCs are reprogrammed following 3DIV with FGF2; the rapid ventralizing effect of FGF2 appears to be restricted to a subset of our starting population, perhaps representing multipotential NPCs rather than more lineage-restricted populations or those expressing particular FGF receptors [81].

Although during prenatal embryogenesis, cortical NPCs do not generate GABAergic neurons [19], postnatally, these cells have been shown to contribute to the adult subventricular zone, where they generate interneurons of the olfactory bulb [82–84]. In addition, postnatally, cortical NPCs also generate oligodendrocytes [85]. Therefore, instead of promoting a ventral fate, FGF2 may be reprogramming these cells to take on a phenotype more characteristic of postnatal cortical NPCs. Indeed, there are cortical NPCs whose development requires FGF2, since FGF2-deficient mice have reduced numbers of cortical neurons [86] and glia [87] and when FGF2 is injected into the embryonic cerebral ventricles, increases in the numbers of neurons and glia are observed [87].

Cortical NPCs Undergo a Glutamatergic to GABAergic Fate Switch Following Exposure to FGF2
Short-term exposure to FGF2 also allows cortical NPCs to generate neurons in the absence of any extracellular signal but changes their phenotype to GABAergic rather than glutamatergic neurons. This contrasts sharply with other studies that have shown that FGF2 induces cortical NPCs proliferation [42] or oligodendrocyte differentiation [88] rather than altering neuronal fate. However, differences in in vitro culture paradigms may explain the apparent conflict. Interestingly, our observed FGF2-induced upregulation of Mash1 and Olig2 is largely independent of SHH signaling, since we still see a significant increase in their expression even in the presence of the SHH antagonist cyclopamine (unpublished observations) and no significant increase in Shh expression (Fig. 3). Despite upregulated expression of ventral TFs in E14 cortical NPCs by FGF2, there is also remarkable heterogeneity of their expression both in the total NPC population and within individual clones. We have also shown that FGF8 induces similar changes in TF expression in cortical NPCs (unpublished observations). It will be interesting to determine the subtypes of GABAergic neurons generated, since there is increasing evidence for different transcription factor requirements for particular subtypes [29, 73].

Recent evidence from the spinal cord has suggested a "molecular code" by which combinatorial expression of many TFs determines neuronal and glial fate decisions [73]. Within the proposed model, Pax6, Ngn2, and Mash1 have key roles in neurogenesis, whereas Olig2 and, again, Mash1 have roles in oligodendrocyte differentiation [73]. By analogy, we hypothesize that combined Pax6 and Mash1 expression (and to some extent Olig2 expression) in cortical NPCs following exposure to FGF2 can give rise to large numbers of GABAergic neurons. In addition, it is known that bipotent, MASH1/OLIG2-positive progenitors in the ventral telencephalon are able to give rise to either GABAergic neurons or oligodendrocytes, depending upon modulation of BMP signaling [37]. If FGF2 reprograms cortical NPCs such that they coordinately express the required levels of Mash1 and Olig2, this could explain our observed rise in GABAergic clones and some clones that contain oligodendrocytes. Furthermore, we observed an FGF2 upregulation of Bmp4 expression after 3DIV and downregulation of its antagonist, noggin (Fig. 3). Therefore, reprogrammed cortical NPCs could be intrinsically bipotent but generate largely GABAergic neurons because of inhibition of oligodendrocyte differentiation by extracellular BMP4.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosure of Potential...
 Acknowledgments
 References
 
Many characteristics of NPCs from different dorsoventral positions of the developing telencephalon are maintained in vitro. However, even short-term exposure of cultured NPCs to exogenous FGF2 leads to a profound and heterogeneous change in their molecular phenotype that has important consequences for their subsequent fate. This has implications for the large-scale generation of homogeneous NPCs designed for therapeutic use and poses many questions as to the signaling pathways by which neural cell fate can be respecified and whether we can predict their behavior on the basis of their molecular phenotype.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Nigel Pringle, Bill Richardson, David Anderson, and Chuck Stiles for the kind gift of antibodies and Noel Buckley for critical reading of the manuscript. This research was supported by grants from the Medical Research Council (MRC) (G9804985), the Biotechnology and Biomedical Sciences Research Council (BBS/B/14736), and the Wellcome Trust (051910). S.E.F. is supported by an MRC studentship. M.F.H. is currently affiliated with Syntaxin Ltd. (Salisbury, U.K.).


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 Results
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 Summary
 Disclosure of Potential...
 Acknowledgments
 References
 

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