Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA
*Author for correspondence (e-mail: aghosh{at}jhmi.edu)
Accepted June 28, 2001
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SUMMARY |
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Key words: Cerebral cortex, Progenitor cells, Cell specification, Cell signalling, Glia, Neuron, Mouse
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INTRODUCTION |
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Stem cells can generate differentiated progeny such as neurons and glia by either cell autonomous or non-cell autonomous mechanisms. For example, a stem cell might undergo asymmetric division to give rise to a stem cell daughter and a daughter cell with a restricted proliferative and differentiation capacity. Alternatively, a stem cell might give rise to uncommitted progeny that are capable of differentiating along several different pathways in response to extracellular signals. The extracellular signal might select for a particular cell type in one of two ways. The signal might act by instructing a multipotent cell to adopt a specific developmental fate (instruction), or it might act as a survival factor for a lineage-committed daughter cell (selection). The sequential generation of neurons and glia in the cerebral cortex offers a good model system for evaluating the instructive and selective effects of extracellular factors, as well as cell autonomous mechanisms, in cell fate specification.
In the rodent cerebral cortex, neurons are generated from embryonic day 12 (E12) through E20, and glia are generated postnatally (Bayer and Altman, 1991). Lineage studies indicate that the developing cortex contains multipotent progenitor cells capable of generating both neurons and glia (Kilpatrick et al., 1995; Luskin et al., 1988; Reid et al., 1995; Walsh and Cepko, 1988; Walsh and Cepko, 1992), although separate precursors for neurons and glia have also been described (Davis and Temple, 1994; Luskin et al., 1993; Luskin et al., 1988; Price and Thurlow, 1988; Williams and Price, 1995). There are several mechanisms that might contribute to the sequential generation of neurons and glia during cortical development. One possibility is that a multipotent stem cell gives rise to neuronal restricted progenitors early in development by asymmetric division, and gives rise to glial restricted progenitors late in development. The switch in cell fate could be regulated by a cell autonomous mechanism, for example by loss of intrinsic factors partitioned unequally during mitosis over time. Alternatively, the fate of cells generated at a particular division could be instructively specified by signals present in the extracellular environment. The presence of a neuronal fate inducing signal early in development and a glial fate inducing signal later in development could then account for the observed temporal pattern of cellular differentiation.
To analyze the influence of extracellular signals in cell fate decisions in the cortex, we have developed a cortical slice overlay assay that allows one to define the spatial and temporal distribution of signals that regulate differentiation without having a prior knowledge of the identity or nature of the signal. By culturing cortical progenitor cells directly on top of cortical slices we can investigate whether the sequential generation of neurons and glia in the cortex is an intrinsic property of the progenitor cells or if their fate is regulated by local environmental signals. Also, by performing clonal analysis on cells growing underneath cortical slices separated by a porous membrane, we can determine the nature of the signal, and whether the signals act instructively to alter cell fates or to selectively promote the survival of lineage restricted precursors. Here we describe experiments that show that the fate of early cortical progenitors can be influenced by local extracellular signals. We also show that a developmentally regulated extracellular signal induces glial cell fates, and that late cortical progenitors are restricted to glial fates. Finally, we show that FGF2 and CNTF influence cell fate and glial differentiation respectively, suggesting that these signals may co-operatively regulate the transition from neurogenesis to gliogenesis in vivo.
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MATERIALS AND METHODS |
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Immunostaining of slice overlay cultures on membranes and identification of cell types
Slices were fixed overnight with 4% paraformaldehyde (PFA) at 4°C and processed for immunofluorescence analysis by blocking nonspecific binding overnight at 4°C with 3% BSA, 0.3% Triton X-100, and 1% goat serum in PBS, followed by a further overnight 4°C incubation with primary antibody in 3% BSA and 0.3% Triton X-100 in PBS, and secondary antibodies in 3% BSA, 0.3% Triton X-100, and 1% goat serum in PBS. For BrdU immunofluorescence analysis slices were fixed overnight in 4% PFA, postfixed for 1 hour in 70% ethanol, permeabilized with 0.4% Triton X-100 in PBS for 1 hour, incubated with 2 N HCl for 30 minutes, 0.1 M NaB2O7 for 30 minutes, followed by overnight blocking, primary antibody incubation, and secondary antibody staining as above. The primary antibodies used were rabbit polyclonal anti-GFP (1:3000, Molecular Probes), mouse monoclonal anti-GFP (1:1000, Molecular Probes), mouse monoclonal anti-MAP-2 (1:3000, Sigma), mouse monoclonal anti-GFAP (1:1000, Sigma), mouse monoclonal anti-BrdU (1:400, Becton Dickinson) and rabbit polyclonal anti-NG2 (1:1000, Chemicon). The fluorescent secondary antibodies were goat anti-rabbit or goat anti-mouse Oregon Green-conjugated IgG (1:600, Molecular Probes), and goat anti-rabbit or goat anti-mouse Cy3-conjugated IgG (1:600, Molecular Probes). Cell nuclei were stained for 2 hours using Hoechst 33258 (1:2000, Molecular Probes). Images were acquired with a Nikon TE300 Eclipse microscope using IPLab Spectrum 3.2 (Scanalytics) or with a LSM 510 Zeiss confocal microscope equipped with helium-neon and argon lasers.
Most cultures at E15+5 days in vitro (DIV) were scored based on MAP-2 and GFP immunofluorescence since we did not find a definitive marker to identify astrocyte precursors (the RC2 antibody did not work well in our cultures), and mature astrocytic markers such as GFAP were not expressed at 5DIV. In these cultures, GFP-positive/MAP-2-positive (GFP+/MAP-2+) cells were scored as neurons. Neurons classified as pyramidal neurons had one major MAP-2-positive tapering process that frequently terminated in an apical tuft. These cells sometimes also had minor basilar dendrites, which were MAP-2 positive, but of finer caliber than the apical dendrite. GFP+/MAP-2-negative (MAP-2-) cells were scored as glial if they had a differentiated astrocytic morphology, and were scored as undefined if they did not have a differentiated morphology. Cultures at E15+10DIV were scored as neurons or astrocytes based on GFP, MAP-2, and GFAP immunofluorescence.
Clonal analysis
Dissociated E15 cortical progenitor cells isolated from heterozygous GFP transgenic mice were diluted 1:1000 with wild-type progenitors from littermates and plated on poly-l-lysine- and laminin-coated glass coverslips in 12-well tissue culture plates (Costar) at 1x106 cells/well. Each well contained 1 ml of NEUROBASAL medium (Gibco) supplemented with glutamine (Gibco), penicillin-streptomycin (Gibco) and B27 (Gibco). GFP expression was used to confirm that the progenitors were completely dissociated and well isolated from each other so that clones could be unambiguously identified. To examine the influence of growth factors on clonal composition, cultures were treated with 10 ng/ml FGF2 (Amgen), 10 ng/ml PDGF BB (Upstate Biotechnology), 10 ng/ml ciliary neurotropic factor (CNTF; Upstate Biotechnology), or 10 ng/ml EGF (Upstate Biotechnology) for the duration of the experiment. To determine if cortical slices produced diffusible signals that could influence clonal composition, cortical slices from E18 and P15 cortex were placed in membrane inserts, and the inserts were placed over glass coverslips in 12 well plates (Biocoat; Becton Dickinson) containing E15 GFP and E15 wild-type mixed cortical cultures as described above.
Immunostaining of dissociated cells on coverslips
Cells were fixed for 15 minutes at room temperature with 4% paraformaldehyde/4% sucrose, followed by blocking nonspecific binding for 2 hours with 3% BSA, 0.3% Triton X-100, and 1% goat serum in PBS. The cells were then incubated with primary antibody overnight at 4°C in with 3% BSA and 0.3% Triton X-100 in PBS, and then with secondary antibody at room temperature for 1 hour in 3% BSA, 0.3% Triton X-100 and 1% goat serum in PBS. The antibodies used were the same as described above.
Data analysis
All of the experiments involving slice overlay cultures were carried out at least three times, and analysis was based on quantification of at least 2 independent experiments with 15-20 slices scored per experimental condition. Approximately 200 cells were scored per slice. The experiments examining the effects of purified growth factors, and analysis of clone number over time were based on quantification of at least two independent experiments with at least 3 wells per experiment. Data are represented as mean ± s.e.m.
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RESULTS |
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While these experiments indicated that factors present within the embryonic and postnatal cortical environment could influence the differentiation of cortical cells, they did not reveal whether these signals act upon dividing progenitor cells or upon newly postmitotic cells. To specifically examine the influence of the cortical environment on dividing progenitor cells, we labeled dividing cells by adding Bromodeoxyuridine (BrdU) to the medium, and subsequently examined the differentiated fate of dividing progenitor cells by double immunofluorescence for BrdU and GFP (in these experiments BrdU was present throughout the culture period). While a vast majority of the GFP+/BrdU+ cells cultured over E18 slices differentiated into neurons (Fig. 2A,D), the converse was true for GFP+/BrdU+ cells cultured over P15 slices. (Fig. 2C,F). The ability of cortical signals to induce neuronal differentiation of progenitor cells was restricted to the embryonic period, since there were virtually no GFP+/BrdU+ neurons in the cortical slice overlay assays in which E15 GFP cells were plated over P0 or P15 cortical slices (Fig. 2B,C,E,F). Thus, the differentiated fate of embryonic progenitor cells can be influenced by developmentally regulated extracellular signals.
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To evaluate the influence of extracellular signals on clone survival and cell fate specification, we analyzed clone viability and clone composition in cultures growing under control conditions (medium) or underneath E18 or P15 cortical slices (Fig. 4A). Our analysis was restricted to GFP clones of two or more cells (indicating that the cells had undergone at least one round of cell division in culture). To determine if factors produced by E18 or P15 slices differentially affected clone viability, we counted the change in the total number of clones over the culture period under different experimental conditions. In control cultures, at 5 days the number of clones was 75±5% of the clones present at day 1. In comparison, clone viability at 5 days underneath E18 and P15 was 67.6±3% and 66.2±2% respectively. Thus, although there is some loss of clones under all conditions, clone survival rates were comparable under E18 and P15 slices.
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Quantification of the total number of each class of clones in different experimental conditions indicated that there was an absolute increase in the number of glial clones when cells were grown below P15 slices (Fig. 4E). This observation, together with the fact that clone viability is comparable under E18 and P15 slices, strongly suggests that a factor produced by the postnatal cortex exerts an instructive influence on cortical progenitors to induce glial fates. At the same time the drop in both the absolute and relative number of neuronal clones under P15 slices suggests that the postnatal factor inhibits neuronal fates. It is also noteworthy that E18 slices induce a 5-fold increase in mixed clones. Since mixed clones arise from multipotent progenitors, this result suggests that E18 cortex may provide a signal that is important in maintaining a stem cell phenotype.
To determine if the embryonic or postnatal cortex exerted a major effect on cell proliferation within clones, we also counted the total number of cells per clone under different experimental conditions. As shown in Fig. 4E the presence of E18 or P15 cortical slices did not significantly affect neuronal clone size. However, culturing cells underneath P15 slices did lead to a small increase in glial clone size, suggesting that the P15 cortex might also produce a mitogen for glial cells.
The strength of our clonal analysis rests on our ability to identify groups of cells as being clonally derived, and the identification of an isolated group of cells as a clone requires that there be limited migration of cells away from the clone. To determine the extent of migration of GFP cells away from a clone, we carried out time-lapse imaging of identified GFP-positive clones over periods up to 3 days in culture. As expected, clone size increased during these observations as a result of cell division, but there was little, if any, migration. An example of a clone imaged between 4 and 7 days in vitro is shown in Fig. 4F and represents the spatially restricted expansion of a typical clone. We also measured the average clone diameter and average distance between nearest neighbor clones to determine the likelihood of clonal overlap. The average clonal diameter (87±6 µm) was much smaller than the distance between nearest neighbor clones (410±26 µm), indicating that the probability of clonal overlap was extremely small.
FGF2 and CNTF regulate distinct aspects of glial cell differentiation
While the clonal analysis indicates that the postnatal cortex produces a signal that can induce glial fates, it does not address whether glial fate specification and terminal differentiation can be independently regulated. One possibility is that once a cell receives a cell fate specification signal, it initiates a cascade of biochemical changes that inevitably leads to terminal differentiation. Alternatively, fate specification and terminal differentiation may be under the control of distinct extracellular signals. One way to distinguish between these possibilities is to ask if purified growth factors that induce glial fates also induce terminal differentiation. We therefore decided to examine the effects of a number of putative glial differentiation signals on cell fate specification and terminal differentiation.
To determine if potential gliogenic signals can induce a cell fate switch in cortical progenitor cells, we cultured E15 GFP cortical progenitor cells in clonal culture in the presence of CNTF, PDGF, EGF, or FGF2 for 5 days, and then analyzed clonal composition. In medium alone, 75% of the clones were neuronal, 10% were mixed, and 15% were glial (Fig. 5). Of the various factors tested, only FGF2 was effective in inducing a cell fate switch comparable to that induced by postnatal cortex (cf. Fig. 4D). In 10 ng/ml FGF2, there was a dramatic reduction in the number of neuronal clones (to 25%), and an increase in the number of glial clones (to 60%). This FGF2 effect is similar to that reported by Temple and colleagues using clonal analysis of isolated cortical progenitors (Qian et al., 1997). In contrast, CNTF, EGF and PDGF did not affect the percentage of glial clones, although EGF led to an increase in mixed clones. The EGF effect on mixed clones is similar to the effect of E18 slices on mixed clones (cf. Fig. 4D), suggesting that EGF may be a component of the E18 activity. Analysis of clone composition and number showed that the shift in clone composition induced by FGF2 cannot be accounted for by selection (data not shown), indicating that FGF2 instructively induces glial fates.
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Finally, we examined the effect of developmentally regulated extracellular signals on the terminal differentiation of glial cells. Very few of the cells expressed oligodendrocyte markers in our cultures, and therefore our analysis was restricted to an analysis of extracellular signals on astrocytic differentiation. To determine if developmentally regulated signals regulate astrocyte differentiation, we analyzed expression of the astrocyte marker GFAP in E15 GFP cells that had been plated over E18 or P15 slices but that had not differentiated into neurons (GFP+/MAP-2- cells). As shown in Fig. 6A-C, after 10 days in culture only 25% of the E15 GFP+/MAP-2- cells plated over E18 slices had differentiated into GFAP+ astrocytes, while the remaining 75% remained GFAP negative, indicating that they had not undergone terminal astrocytic differentiation. In contrast, over 70% of the E15 GFP+/MAP-2- cells plated over P15 cortical slices differentiated into GFAP+ astrocytes (Fig. 6A-C). Thus the terminal differentiation of cortical progenitor cells into GFAP+ astrocytes can be regulated by inductive signal that is produced by the postnatal cortex.
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DISCUSSION |
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The observation that postnatal cortical progenitors are restricted to glial fates suggests that there may be a developmental restriction of cell fate in the cortical progenitor population. While the alternative possibility, that postnatal cortical progenitors are not derived from embryonic cortical progenitors, cannot formally be excluded, it is worth considering how cortical progenitors might undergo cell fate restriction during development. Our findings suggest that cell fate restriction may be a consequence of extracellular signals acting on a multipotent progenitor. This possibility is supported by several of our observations. First, we find that embryonic cortical progenitor cells can differentiate into glia when placed onto postnatal cortical slices without first generating neurons. Thus the progenitors do not necessarily generate neurons before generating glia as would be predicted if cells undergo a cell autonomous switch. Second, our experiments on the influence of late cortical slices on clonal composition indicate that a cortical signal can induce progenitors to adopt glial fates at the expense of neuronal fates. Third, we find that there is a developmental increase in an extracellular signal that induces glial cell fates. Together these observations suggest that an extracellular signal acts on a multipotent progenitor to induce a glial fate. The response of older progenitors placed on embryonic slices suggests that once cortical progenitors adopt a glial fate, they cannot give rise to neurons even in a neurogenic environment. Such a restriction of progenitor cells to glial fates late in development is similar to a developmental fate restriction in neuronal subtype that has been reported by McConnell and colleagues (McConnell, 1988; Frantz and McConnell, 1996).
Our clonal analysis indicates that a diffusible signal, present in postnatal cortex, can induce glial cell fates. To begin to identify the molecular components of this signal we tested a number of growth factors that are expressed in the developing cortex. The only factor that induced a cell fate switch comparable to that induced by postnatal cortex was FGF2. This adds to the growing evidence that FGF2, which is expressed in the embryonic cortex, can regulate both cell proliferation and cell fate specification in the cortex (Ghosh and Greenberg, 1995; Qian et al., 1997). Injection of FGF2 into the embryonic cortex leads to an increase in the number of neurons and glia, and fgf2 null mice have reduced numbers of neurons and glia (Dono et al., 1998; Ortega et al., 1998; Vaccarino et al., 1999). These in vivo results have been interpreted as reflecting a mitogenic role for FGF2, but they are also consistent with a role for FGF2 in glial fate specification. It will be of interest to carefully examine the fgf2 null mice to determine if the effects in vivo are principally due to effects on proliferation, or cell fate specification.
Our data also suggest that factors present in the postnatal cortex act to promote the terminal differentiation of astrocytes. CNTF appears to be a component of the postnatal signal that promotes astrocyte differentiation since CNTF is expressed in the postnatal cortex. Also, adding CNTF to E18 slices induces GFAP expression in both the GFP cells plated over the slice, and in cells within the slice (T. M. and A. G., unpublished observations). These observations add to the evidence that CNTF is an important regulator of astrocyte differentiation (Barres and Raff, 1994; Bonni et al., 1997; Johe et al., 1996; Mi and Barres, 1999). It is striking that CNTF is unable to induce a cell fate switch, although it is very effective in inducing terminal astrocytic differentiation. One possibility is that FGF2 induces CNTF responsiveness in cortical progenitor cells. That would provide for a molecular mechanism by which a cell fate specification signal might induce competence for terminal differentiation in a progenitor cell.
In our slice overlay cultures, cortical progenitor cells typically did not differentiate into oligodendrocytes, the other major class of glial cells. Also, none of the factors we tested was effective in inducing oligodendrocyte differentiation. While this may reflect the existence of other oligodendrocyte differentiation signals (Barres and Raff, 1994; Park et al., 1999), it may also reflect the limitation of the cortical slice overlay assay, which has only been examined for 10 days in vitro. We have previously shown that cortical progenitors grown in FGF2 will differentiate into oligodendrocytes after 10 days in vitro (Ghosh and Greenberg, 1995), and it will be useful to know if cells plated over slices behave similarly in longer term cultures.
Our observations suggest the following model for cell fate specification in the cortex (Fig. 7). During the neurogenic period the cortical progenitor is a multipotent cell that adopts a neuronal fate unless it is exposed to a glial fate-inducing signal. FGF2 can act as a mitogen and a glial fate-inducing factor for this cell, but during the neurogenic period most progenitors exit the cell cycle and differentiate into neurons because of low levels of extracellular FGF2. A developmental increase in FGF2 signaling (possibly in cooperation with other molecules) induces a glial fate on the remaining dividing progenitors such that all subsequent progeny are restricted to becoming glial cells. These cells, however, do not undergo terminal differentiation unless they are exposed to a second signal that specifically regulates terminal differentiation. CNTF is likely to be an important component of a terminal astrocyte differentiation signal. Such a mechanism, in which developmentally regulated extracellular factors induce multipotent progenitors to adopt a specific cell fate, and subsequently to induce terminal differentiation, might mediate the sequential generation of neurons and glia throughout the nervous system.
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ACKNOWLEDGMENTS |
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