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The Journal of Neuroscience, December 1, 1999, 19(23):10383-10389
Bipotent Cortical Progenitor Cells Process Conflicting Cues for
Neurons and Glia in a Hierarchical Manner
John K.
Park1, 2,
Brenda
P.
Williams3,
John A.
Alberta2, and
Charles D.
Stiles2
1 Division of Neurosurgery, Brigham and Women's
Hospital and 2 Department of Microbiology and Molecular
Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 02115, and 3 Department of Molecular
Pathology, University College London Medical School, The Windeyer
Institute of Medical Science, W1P 6DB London, UK
 |
ABSTRACT |
Neurons and glia of the cerebral cortex are thought to arise from a
common, multipotent progenitor cell that is instructed toward alternate
fates by extracellular cues. How do these cells behave when confronted
with conflicting cues? We show here that nestin-positive
neuroepithelial (NE) cells from embryonic day 14 rat cortex
coexpress surface receptor proteins for ciliary neurotrophic factor
(CNTF) and platelet-derived growth factor (PDGF). Both sets of these
receptor proteins are functional in NE cells, as shown by
ligand-dependent activation of downstream signal-generating proteins.
Transient (30') exposure to CNTF instructs NE cells toward an astrocyte
fate. Brief exposure to PDGF initiates neuronal differentiation.
However, when challenged with conflicting cues, PDGF is dominant to
CNTF. Moreover, CNTF-treated NE cells can be "redirected" by a
subsequent exposure to PDGF to form neurons instead of astrocytes,
whereas the converse is not true. The asymmetric relationship between
CNTF and PDGF indicates that these two growth factors act on a common
progenitor cell that has, at a minimum, two fates available to it
rather than separate populations of precommitted neuroblasts and
astroblasts. This bipotent progenitor cell processes conflicting cues
for neurons and glia in a hierarchical manner.
Key words:
neurons; astrocytes; CNTF; PDGF; cortical development; neuroepithelial cells; signal transduction
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INTRODUCTION |
Histogenetic studies of the rat
brain have contributed greatly to our current understanding of
mammalian cerebral cortical development (Bayer and Altman, 1991 ).
During embryonic days 13 and 14 (E13-E14) of a 21 d
gestational cycle, neuroepithelial (NE) cells expressing the
intermediate filament nestin (Hockfield and McKay, 1985) undergo cell
division within the ventricular zone of the developing brain. At E16,
the subventricular germinal zone develops, and postmitotic neurons
start to appear in both the ventricular and subventricular areas. These
neurons migrate along radial glia cells to their eventual destinations
within the deeper cortical layers from E17 to E21. At E19, the mostly depleted ventricular zone stops producing neurons, whereas the subventricular zone continues to generate neurons and now also glial
cells. The subventricular zone neurons migrate to form the superficial
layers of cortex from E21 to postnatal day 3. From E21 on, the
subventricular zone is thought to produce only glial cells.
The cell of origin for both neurons and glia is thought to be a common,
multipotent progenitor cell. Transplantation studies show that the fate
choices of this progenitor cell can be dictated by extracellular cues
in the local environment (Brustle et al., 1998; Flax et al., 1998).
These fate choices are obviously mutually exclusive. A fully
differentiated cell can be a neuron, an astrocyte, or an
oligodendrocyte, but not a combination of the two or three lineages.
Given the unlikelihood that the extracellular signals controlling fate
choice can be strictly compartmentalized in space and time within the
developing embryo, a question arises. How do multipotent neural
progenitor cells process conflicting cues?
This question can be addressed in vitro using
undifferentiated, nestin-positive NE cells excised from the ventricular
zone of E14 rats. These NE cell cultures give rise to neurons,
astrocytes, or oligodendrocytes in response to specific growth factors.
Platelet-derived growth factor (PDGF) for example initiates neuronal
differentiation, whereas (under varying conditions of cell culture)
bone morphogenic proteins, basic fibroblast growth factor (bFGF), or
ciliary neurotrophic factor (CNTF) induce astrocyte development, and
thyroid hormone stimulates oligodendrocyte development (Gross et al.,
1996; Johe et al., 1996; Bonni et al., 1997; Williams et al., 1997). In
principle, NE cell cultures can be composed of unipotent progenitor
cells that each generate a single cell type, bipotent progenitor cells that generate restricted pairs of cell types, and/or multipotent progenitor cells that give rise to all three cell types. In the experiments summarized here, we show that NE cell cultures taken from
the ventricular zone of E14 rats contain an abundant population of
cells that are, at a minimum, developmentally bipotent. These bipotent
NE cells process conflicting cues to form neurons and astrocytes in a
hierarchical manner. The data provide a possible explanation for why
neurons appear before astrocytes in the developing mammalian brain.
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MATERIALS AND METHODS |
Cell cultures. The cerebral cortices of Wistar Furth
(Harlan, Madison, WI) rat E14 embryos were dissected in serum-free
medium (DMEM supplemented with glucose, transferrin, insulin, selenium, progesterone, thyroxine, tri-iodothyronine, putrescine, and bovine serum albumin) (Williams et al., 1991; Williams and Price, 1995) and
dissociated into a single cell suspension by trituration through 21 and
23 gauge hypodermic needles. The cells were centrifuged and resuspended
in serum-free medium before plating on
poly-D-lysine-coated 12 mm glass coverslips
(Fisher Scientific, Pittsburgh, PA) placed in 24-well tissue plates
(Falcon, Lincoln Park, NJ). The initial plating densities were 2 × 105 cells per coverslip in a volume of
50 µl. After allowing 30 min for the cells to settle and attach to
the coverslips, 0.45 ml of additional serum-free medium was added to
each well. For clonal analysis experiments, cultures were infected at
this time with the BAG retroviral vector that encodes the enzyme
 -galactosidase (-gal) (Price et al., 1987). The following
day, cultures were stimulated with CNTF (Upstate Biotechnologies, Lake
Placid, NY), PDGF (Upstate Biotechnologies), or solvent controls as
indicated, washed three times with serum-free medium, and maintained in
serum-free medium. For experiments in which the cells were stimulated
simultaneously, CNTF (30 ng/ml) and PDGF B-B (The homedimeric B-B
isoform of PDGF) (30 ng/ml) were present for 4 hr and then washed out
three times with serum-free medium. In the sequential stimulation
experiments, cultures were exposed to the first growth factor for 4 hr,
washed three times with serum-free medium, exposed to the second growth factor for 4 hr, again washed three times with serum-free medium, and
then maintained in serum-free medium. Thereafter, all cultures were fed
every 2 d with serum-free medium, and cell staining and immunoblotting analyses were performed at the indicated times. In some
experiments, NE cell cultures were first exposed to 15 µg/ml
5,6-dichloro--D-ribofuranosyl-benzimidazole
(DRB) (Sigma, St. Louis, MO), a reversible inhibitor of RNA synthesis
alone for 1 hr before stimulation with CNTF (30 ng/ml) or control
solvent for 4 hr in the continued presence of DRB. The DRB with or
without CNTF was removed by washing three times with serum-free
medium after the 4 hr growth factor stimulation period.
Immunofluorescence. To determine the presence of growth
factor receptors on NE cells, cells plated on glass coverslips as described above were fixed with 4% paraformaldehyde and incubated with
primary antibodies (Abs) against nestin (Developmental Studies Hybridoma Bank, Iowa City, IA) (Hockfield and McKay, 1985), the CNTF
receptor (CNTF-R) (Santa Cruz Biotechnology, Santa Cruz, CA), and the
PDGF receptor (PDGF-R) (Santa Cruz Biotechnology). After washing, cells
were incubated with species and isotype-specific aminomethylcoumarin
acetate (AMCA)-, indocarbocyanine (Cy3)-, or cyanine (Cy2)-conjugated
secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and
viewed using a Nikon (Tokyo, Japan) Optophot-2 microscope. Control
coverslips were also fixed with 4% paraformaldehyde but incubated only
with the species and isotype-specific Cy3- or Cy2-conjugated secondary
antibodies. When viewed using a Nikon Optophot-2 microscope, there was
no cell-associated fluorescence. To demonstrate the signaling ability
of these growth factor receptors, NE cells were stimulated for 10 min
with either CNTF or PDGF before fixation with 4% paraformaldehyde.
They were then stained with primary antibodies against nestin and
either phosphorylated signal transducer and activator of transcription
(STAT 3) (gift of D. Frank, Dana-Farber Cancer Institute,
Boston, MA) or phosphorylated mitogen-activated protein (MAP)
kinase (Promega, Madison, WI), respectively. After washing, cells were
incubated with the secondary antibodies noted above. Determination of
cell phenotypes was performed as described previously (Williams et al.,
1991; Williams and Price, 1995). Neurons were identified using the
monoclonal antibody (mAb) TuJ1 (Geisert and Frankfurter, 1989) and
confirmed by staining with the monoclonal antibodies
anti-neurofilament 68, 160, or 200 kDa (Sigma), MAP2
(Sigma), or anti-neuron synaptosomal-associated protein
(SNAP-25) (specific for a 25 kDa neuron synaptosomal-associated protein) (Transduction Laboratories, Lexington, KY). Astrocytes were
identified using anti-GFAP monoclonal antibodies (Sigma) (Bignami et
al., 1972), and oligodendrocytes were identified using the O4 antibody
(Boehringer Mannheim, Indianapolis, IN) (Sommer and Schachner, 1981).
NE cells were identified as cells that stained with an anti-nestin
antibody but not with antibodies that recognize differentiated cell
types. Determination of clones was performed using rabbit
anti--galactosidase serum (Williams et al., 1991), and NE clones are
defined as clones in which all lacZ-positive cells are also positive
for nestin and negative for all other markers. Likewise, astrocyte and
neuronal clones are defined as clones in which all lacZ-positive cells
are also positive for GFAP or TuJ1, respectively, and negative for all
other markers. In some experiments, total cells were scored rather than
-gal-positive clones. When scoring total cells, the percentage of
GFAP-positive astrocytes or TuJ1-positive neurons was determined by
counting five fields per coverslip under a fluorescent microscope using a 63× objective. For both clonal analysis and total cell analysis, the
significance of the increase in astrocytes or neurons observed was
calculated using the Bonferroni-Dunn procedure.
Immunoblots. E14 cell cultures were stimulated with CNTF and
lysed at the specified time intervals with NP-40. For determination of
GFAP expression, lysates were size fractionated on an
SDS-polyacrylamide gel, transferred to Immobilon-P membranes
(Millipore, Bedford, MA), and immunoblotted with anti-GFAP monoclonal
antibodies. For determination of signal transduction molecule
activation, lysates were immune precipitated with an
anti-phosphotyrosine mAb (4G10) (Upstate Biotechnologies), size
fractionated on an SDS-polyacrylamide gel, transferred to Immobilon-P
membranes and immunoblotted with anti-gp130 (Upstate Biotechnologies),
anti-STAT 1 (gift of D. Frank), or anti-STAT 3 Abs. All antibody
signals were visualized with ECL (Amersham, Arlington Heights, IL)
and/or quantitated with Attophos (JBL Scientific, Inc., San Luis
Obispo, CA) using a fluorimager apparatus (Molecular Dynamics,
Sunnyvale, CA). In some experiments, NE cell cultures were also
stimulated with CNTF in the absence or presence of DRB and similarly
processed as described.
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RESULTS |
Coordinate expression of functional receptors for CNTF and PDGF in
cultured NE cells
Cells isolated from the developing cerebral cortices of E14 rats
were plated and cultured in serum-free medium overnight. Immunofluorescence staining (Fig. 1)
shows that >75% of the total cells in these cultures express the
intermediate filament nestin but are negative for markers found on
differentiated cell types and are thus identifiable as NE cells. More
than 90% of the NE cells express surface receptors for both CNTF and
PDGF (Fig. 1). These CNTF and PDGF receptors are uniformly
ligand-responsive and functional. This can be shown by exposing the
cells to CNTF or PDGF and monitoring the activation of downstream
signal-generating proteins with phosphopeptide-directed antibodies.
CNTF is thought to induce cellular responses primarily (though not
exclusively) through activation of JAK-STAT signal transduction
pathways (Bonni et al., 1997). Conversely, PDGF signals primarily
(though again, not exclusively) via the Ras-Raf-MAP kinase pathway
(Schlessinger and Ullrich, 1992). Accordingly, we stimulated NE cells
with either CNTF or PDGF for 10 min and then conducted
immunofluorescent staining with antibodies directed against the
phosphorylated, activated forms of either STAT 3 or MAP kinase,
respectively. As shown in Figure 2, both
CNTF and PDGF activate downstream signaling proteins in the majority of
NE cells. Collectively, the images in Figures 1 and 2 show that
functional receptors for CNTF and PDGF are coordinately expressed in
cultured NE cells.
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Figure 1.
Neuroepithelial cells express both CNTF and PDGF
receptors. One day after plating on glass coverslips, E14 cultures were
fixed with 4% paraformaldehyde and then immunostained with antibodies
to nestin, CNTF receptor, or PDGF receptor. Secondary antibodies
conjugated to AMCA, Cy2, and Cy3, respectively, were used to visualize
the target molecules.
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Figure 2.
CNTF-R and PDGF-R expressed on NE cells are
functional. E14 cells plated on glass coverslips were stimulated with
CNTF (30 ng/ml) or PDGF (30 ng/ml) and stained with antibodies directed
against nestin and either activated STAT-3 or MAP kinase, respectively.
Secondary antibodies conjugated to AMCA, Cy2, and Cy3, respectively,
were used to visualize the target molecules.
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Transient activation of CNTF receptors initiates astrocyte
differentiation in NE cell cultures
In previous studies, we reported that even transient (30')
exposures to PDGF can induce neuronal differentiation in E14 NE cell
cultures. Although outward signs of neuronal development do not appear
until several days after PDGF treatment, the sustained developmental
response does not reflect persistently activated PDGF receptors. This
is demonstrated by the ability to uncouple receptor activation from
neuronal development by a reversible inhibitor of RNA (Williams et al.,
1997). These central features of the response to PDGF are seen also in
the astrocytic differentiation of NE cells in response to CNTF. In
accordance with previous studies using bFGF-expanded NE cell cultures
(Johe et al., 1996), nanomolar concentrations of CNTF can promote
astrocyte differentiation in primary, nonexpanded cell populations as
well (Fig. 3). Outward signs of astrocyte
formation are not detectable until 3-4 d after exposure to CNTF (Fig.
4), but CNTF stimulation periods as brief as 30 min are sufficient to initiate this response (Fig.
5).
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Figure 3.
CNTF induces the expression of GFAP. A
dose-response curve for astrocyte formation was established by
exposing NE cultures to increasing concentrations of CNTF for 4 hr.
Expression of GFAP in cell cultures was assessed by immunoblotting
5 d later using an anti-GFAP monoclonal antibody.
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Figure 4.
CNTF induces the differentiation of astrocytes. A
time course for the developmental response to CNTF was determined by
exposing NE cells to CNTF (30 ng/ml) for 4 hr. On the following 5 consecutive days, coverslips were stained with an anti-GFAP mAb, and
the percentages of GFAP-positive cells were determined. *
denotes 0.
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Figure 5.
Transient exposures of CNTF are sufficient to
induce astrocyte differentiation. The minimum time interval needed for
NE cells to become instructed by CNTF to form astrocytes was determined
by exposing cultures to CNTF (30 ng/ml) for increasing amounts of time.
At the indicated times, CNTF was removed, and cultures were washed with
serum-free medium. GFAP expression was assessed after 5 d of
culture in serum-free medium. The number of GFAP-positive cells per
field under a 63× objective as defined by immunostaining is
shown.
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As with PDGF, the long-term developmental response to a brief pulse of
CNTF cannot reflect persistently activated downstream signaling
molecules. Activation of the CNTF receptor subunit gp130 and downstream
signal generating proteins STAT 1 and STAT 3 is relatively transient,
even in the continued presence of CNTF (Fig. 6). Moreover, receptor activation and
signal transduction can be uncoupled from the developmental response by
a reversible inhibitor of RNA synthesis, DRB. Cells treated with DRB
before, and during the time of, CNTF stimulation fail to develop into
astrocytes, even when cultures are maintained for up to 2 weeks (Fig.
7A). Immunoblot studies show
that DRB has no effect on the activation of gp130, STAT 1, and STAT 3 by CNTF (Fig. 7B), nor does DRB act as an irreversible
poison of astrocyte formation. Cells treated with DRB, but washed
before subsequent stimulation with CNTF alone, develop into astrocytes
as expected (data not shown). This is in keeping with previously
published results on the effects of DRB. When used at a concentration
of 15 µg/ml as in these experiments, DRB inhibits total RNA synthesis
by 66%. Total reversal of its effects occurs within 15 min of its
withdrawal (Smith and Stiles, 1981). The simplest interpretation of the
data in Figures 3-7 is that short exposures to CNTF commit NE cells to
differentiate into astrocytes as a result of downstream signaling
events that require new RNA synthesis.
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Figure 6.
CNTF activates gp130 and STATs in NE cells. NP-40
lysates of CNTF (30 ng/ml) stimulated NE cell cultures were made at the
times indicated. The lysates were then immune precipitated with an
anti-phosphotyrosine mAb (4G10), size fractionated on an
SDS-polyacrylamide gel, and immunoblotted with anti-gp130, anti-STAT 1, and anti-STAT 3 mAbs.
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Figure 7.
Differentiation of astrocytes is
transcription-dependent. A, NE cell cultures were
stimulated with CNTF (30 ng/ml) for 30 min in the absence or presence
of DRB, a reversible inhibitor of RNA synthesis, as described
previously (Williams et al., 1997). After 4 d incubation in
factor-free medium, cells were assessed for GFAP expression using mAb.
The percentage of GFAP-positive cells per culture is shown. * denotes
0. B, As controls for the experiment in
A, NE cell cultures were stimulated with CNTF (30 ng/ml)
for 15 min in the absence or presence of DRB. Cell lysates were then
processed and immunoblotted with anti-gp130, anti-STAT 1, and anti-STAT
3 mAbs.
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The PDGF cue for neuronal differentiation is dominant to the CNTF
cue for astrocyte differentiation
As shown in Figures 1 and 2, CNTF and PDGF receptors are
coordinately expressed and functional on virtually all NE cells. We
therefore examined the effects of simultaneous and sequential growth
factor stimulation on single NE cells using a retroviral vector that
encodes the enzyme -gal. Infection of cells with a limiting titer of
retroviral particles allows for the subsequent identification of cell
populations derived from an individual clone. As shown in Table
1 (columns 1-6), the total number of clones in control, or growth factor-treated, cultures is essentially constant. Thus the factors do not affect the viability of the cells in
our clonal analysis. The results of exposure to CNTF or PDGF singly are
shown in Table 1 (columns 2 and 3). Relative to untreated cultures
(column 1), transient exposure to CNTF converts ~70% of the NE
clones into colonies that contain astrocytes (p = 0.001). Transient exposure to PDGF converts ~80% of the NE clones into colonies that contain neurons (p = 0.005).
Superficially, the data in columns 1-3 would indicate that CNTF and
PDGF act on a common progenitor cell that is, at a minimum, developmentally bipotent. The alternative possibility, that CNTF and
PDGF induce precommitted astroblasts and neuroblasts to complete their
developmental programs, is arithmetically untenable because the overlap
of these two cell types would account for greater than 100% of the
original starting population. At a minimum, between 20 and 30% of the
NE cells would have to be developmentally bipotent to generate the data
in columns 1-3 of Table 1. A less likely but conceptually feasible
explanation of these results is that CNTF activates precommitted
astroblasts and kills precommitted neuroblasts, whereas PDGF does the
opposite. To evaluate this possibility, we exposed cells simultaneously
and also sequentially to the two growth factors (Table 1, columns
4-6). When cells are exposed simultaneously to CNTF and PDGF for time
periods sufficient to commit them to either astrocytes or neurons,
mostly neurons are formed. Additionally, cells that have been exposed
initially to CNTF can be redirected to form neurons instead of
astrocytes if they are exposed subsequently to PDGF. The converse does
not hold. The asymmetry of the response to CNTF and PDGF indicates that
the clonal analysis data in columns 1-3 of Table 1 cannot reflect
offsetting developmental and cytotoxic responses for two different
kinds of cells. Assuming that cells infected by the -gal retrovirus
are representative of the population at large, the competition and
order-of-addition experiments indicate that at least 20-30%, and
likely more, of the NE cells have alternate fates available to them
that can be initiated by CNTF or PDGF. Oligodendrocytes as determined
by O4 and galC immunostaining are infrequently present in these
cultures (data not shown).
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DISCUSSION |
Intrinsic genetic programs and extracellular growth factors are
both necessary for the development of the cerebral cortex. We and
others have used culture systems in which embryonic ventricular zone
cells are harvested and grown in chemically defined, serum-free media.
Through manipulation of both the concentration and timing of growth
factor addition and withdrawal, the role of extracellular signals in
cortical development can be studied (Gage et al., 1995).
Although the culture systems used by various authors have differed with
regard to the species, anatomic location, and developmental age of the
harvested tissue, a common theme has been the addition of either bFGF
or epidermal growth factor (EGF) to the growth media. These growth
factors function as initial mitogenic agents to expand the population
of primary cortical progenitor cells (Gensburger et al., 1987; Cattaneo
and McKay, 1990; Reynolds et al., 1992; Kilpatrick and Bartlett, 1993;
Ghosh and Greenberg, 1995; Gross et al., 1996). The additional effects
of bFGF and its interaction with other growth factors have however
varied across, and sometimes even within, the studies published to date (Kilpatrick and Bartlett, 1993; Baird, 1994; Temple and Qian, 1995;
Vicario-Abejon et al., 1995). EGF has likewise been shown to have
pleiotropic effects on developing cells (Ferri and Levitt, 1995; Ferri
et al., 1996; Kilpatrick and Bartlett, 1993, 1995). The cultures
described here were explicitly propagated in the absence of bFGF and
EGF to avoid their potential confounding effects on the two growth
factors of interest, CNTF and PDGF. In doing so, we also obtained a
starting cell population representative of that found in
vivo rather than one enriched for a mitotically active
subpopulation of cells.
Examination of the NE cells with monoclonal antibodies reveals that
they all express receptors for PDGF as well as CNTF. To determine
whether the receptors are functional, we stimulated cells with either
PDGF or CNTF. PDGF stimulation leads to the activation of MAP kinase
within the NE cells. CNTF stimulation is not as effective in activating
MAP kinase (data not shown) but does induce the rapid and short-lived
activation of the signal transduction molecules gp130 and STAT 1 and
STAT 3. In contrast, previous studies by Rajan and McKay (1998) have
shown that CNTF treatment leads to the prolonged activation of the
JAK-STAT pathway (8 d). A possible explanation for the discrepancy is
the difference between the two culture systems in use. Although both
systems begin with E14 cells, the cells used by Rajan and McKay are
first expanded and passaged in bFGF-containing media. The potential modulating role of bFGF in JAK-STAT signaling was not addressed in
their paper. The cells in our study are stimulated in the absence of
bFGF and on the day after plating.
Through a transcription-dependent mechanism, a brief pulse of CNTF
induces the differentiation of NE cells into astrocytes. PDGF
stimulation, also through a transcription-dependent mechanism, leads to
the induction of neuronal differentiation. When CNTF and PDGF are
presented to NE cells simultaneously or in succession, PDGF is dominant
to CNTF and can override its effects as well. The target NE cells
therefore appear to be, at a minimum, bipotent. Another finding of the
clonal analysis experiments is that, although a vast majority of the NE
cells are undifferentiated at the time of harvesting (as defined by
their expression of nestin), not all of them are necessarily
uncommitted. In fact, approximately one-third of the cells appear to be
committed to a neuronal fate in the absence of in vitro PDGF
stimulation, reflecting perhaps their previous in vivo
exposure to PDGF (Williams et al., 1997). The apparent heterogeneity of
NE cells, even within the ventricular zone itself, may explain why
subtle differences in timing of tissue harvest and culture conditions
have led to disparate results by various research groups.
One concern regarding results obtained using relatively specific
in vitro conditions is their relevance to in vivo
processes. The experiments described here demonstrate that PDGF and
CNTF can act as growth factors to induce the development of neurons and
astrocytes, respectively. This in itself has now been shown by
ourselves and others in three different culture systems (Johe et al.,
1996; Bonni et al., 1997; Williams et al., 1997), supporting its
congruity to in vivo events. An obvious next question is how does the developing cortex reconcile these conflicting cues and form
first neurons then glia? Qian et al. (1997) have hypothesized that the
default fate of NE cells is neuronal, and the development of glia
requires exposure to additional signals. Our results do not support
this hypothesis because the formation of a significant number of
neurons requires the presence of PDGF, and PDGF itself can override the
CNTF-driven development of astrocytes. The readily apparent
explanations for the differing results are the use of rat versus murine
cells and the exclusion of bFGF from our cultures for the reasons given
above. Burrows et al. (1997) have also addressed this question
and report that the timing of progenitor cell maturation is regulated
by developmental changes in EGF receptor (EGFR) expression levels.
Ventricular zone cells initially express low levels of EGFR and respond
to EGF by differentiating into neurons. During later stages of
development, cells (strictly speaking, now part of the subventricular
zone) divide and differentiate into astrocytes in response to EGF
(Burrows et al., 1997). Because our cultures do not contain any EGF, it
is difficult to compare and contrast our results with theirs. A
conclusion that may however be drawn is that there are likely multiple
routes to astrocyte differentiation within the CNS (Rajan and McKay,
1998).
The competing and dominant effects of PDGF over CNTF on determining
cell fate suggest an alternative explanation for the temporal sequence
of brain development in vivo. The intrinsic developmental sequence may instead be hardwired into multipotent neural progenitors at the level of competing growth factor signaling pathways. Induction of astrocyte formation by CNTF is channeled primarily through the
JAK-STAT signaling pathway (Bonni et al., 1997), whereas pathways responsible for neuron formation in PDGF-treated cultures have yet to
be defined. In general, however, cellular responses to receptor
tyrosine kinases such as PDGF draw more heavily on Ras-dependent signaling pathways (Schlessinger and Ullrich, 1992). It is possible that, during development in vivo, STAT-dependent cues for
astrocyte formation cannot be acted on until Ras-dependent signals for
neuron formation have been removed. Consistent with this, developmental studies of the rat brain have shown decreases in subventricular zone
PDGF expression after E19 (Sasahara et al., 1992), the time at which a
significant number of cortical astrocytes begin to appear. Ongoing
studies are directed at better defining the signal transduction
mechanisms regulating the orderly development of the cerebral cortex.
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FOOTNOTES |
Received June 14, 1999; revised Sept. 15, 1999; accepted Sept. 21, 1999.
This work was supported by National Institutes of Health Grant PO1
HD24826-10. J.K.P. was supported by a fellowship from the American
Brain Tumor Association. B.P.W. received support from the University of
London, Central Research Fund, University College London Graduate
School, and the Royal Society. We gratefully acknowledge Stephan
Muhlebach for technical assistance and Dr. David Frank for the gift of
phosphorylation state-specific antibodies to STAT 1 and STAT 3. In
compliance with Harvard Medical School Guidelines on possible conflict
of interest, we disclose that C.D.S. has a consulting relationship with
Upstate Biotechnology and Novartis Pharmaceuticals, Inc.
Drs. Park and Williams contributed equally to this work.
Correspondence should be addressed to Charles D. Stiles, Dana-Farber
Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail:
charles_stiles{at}dfci.harvard.edu.
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Copyright © 1999 Society for Neuroscience 0270-6474/99/192310383-07$05.00/0
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ABSTRACT
INTRODUCTION
