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The Journal of Neuroscience, August 15, 1999, 19(16):7077-7088
Multiple Roles of Bone Morphogenetic Protein Signaling in the
Regulation of Cortical Cell Number and Phenotype
Peter C.
Mabie,
Mark F.
Mehler, and
John A.
Kessler
Departments of Neurology and Neuroscience and the R. F. Kennedy Center for Research in Mental Retardation and Human
Development, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
Members of the bone morphogenetic protein (BMP) family have been
implicated in multiple aspects of neural development in both the
CNS and peripheral nervous system. BMP ligands and receptors, as
well as the BMP antagonist noggin, are expressed in the developing cerebral cortex, making the BMPs likely candidates for regulating cortical development. To define the role of these factors in the developing cerebral cortex, we examined the effects of BMP2 and BMP4 on
cortical cells in vitro. Cells were cultured from
embryonic day 13 (E13) and E16 rat cerebral cortex in the absence or
presence of different concentrations of fibroblast growth factor 2, a
known regulator of cortical cell proliferation and differentiation. At
E13, the BMPs promoted cell death and inhibited proliferation of
cortical ventricular zone cells, resulting in the generation of fewer
neurons and no glia. At E16, the effects of the BMPs were more complex.
Concentrations of BMP2 in the range of 1-10 ng/ml promoted neuronal
and astroglial differentiation and inhibited oligodendroglial
differentiation, whereas 100 ng/ml BMP2 promoted cell death and
inhibited proliferation. Addition of the BMP antagonist noggin promoted
oligodendrogliogenesis in vitro, demonstrating that
endogenous BMP signaling influences the differentiation of cortical
cells in vitro. The distribution of BMP2 and noggin
within the developing cortex suggests that local concentrations of
ligands and antagonists define gradients of BMP signaling during
corticogenesis. Together, these results support the hypothesis that the
BMPs and their antagonist noggin co-regulate cortical cell fate and morphogenesis.
Key words:
bone morphogenetic protein; embryogenesis; fibroblast
growth factor; gliogenesis; neurogenesis; noggin; subventricular zone; ventricular zone
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INTRODUCTION |
All of the major cellular elements
of the brain arise from specialized generative zones, the ventricular
and subventricular zones (VZ/SVZ), that are derived from the
pseudostratified neuroepithelium of the neural tube (Altman and Bayer,
1995 ). The mechanisms that regulate neuroepithelial cell
diversification and the appropriately timed differentiation of neurons
and glia during corticogenesis are poorly understood. Although there is
clearly heterogeneity among the VZ/SVZ populations of cells,
substantial evidence indicates that a proportion of these cells are
multipotential, i.e., they can generate all phenotypes depending on the
cellular microenvironment (Reynolds and Weiss, 1992; Reynolds et al.,
1992; Kilpatrick and Bartlett, 1993; Davis and Temple, 1994; Williams
and Price, 1995; Johe et al., 1996; for review, see Stemple and
Mahanthappa, 1997). Although these cells have the potential to generate
these different neural phenotypes throughout development, neurons,
radial glia, astrocytes, and oligodendroglia are primarily generated in
different temporospatial waves during the embryonic and postnatal
periods. This may reflect changes in the factors to which progenitor
cells are exposed at various stages of development, changes in the
properties of these cells during ontogeny, or both. Further
complicating our understanding of lineage development within the
cortex, there is now increasing recognition that apoptosis is a
prominent and highly regulated event during corticogenesis (Finlay and
Slattery, 1983; Ferrer et al., 1992; Wood et al., 1992; Blaschke
et al., 1996; Takahashi et al., 1996a,b; Furuta et al., 1997; Price et al., 1997). Consequently, selective survival of different populations of progenitor cells may be important not only for the timely
specification of cellular phenotypes but also for the morphogenesis of
the brain.
There is increasing evidence that multiple classes of extracellular
signals regulate the proliferation and differentiation of neural
progenitor cells. Mitogenic factors for neural progenitors include
fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF).
FGF2 regulates the proliferation of cortical VZ progenitor cells
(Murphy et al., 1990; Kilpatrick and Bartlett, 1995; Johe et al., 1996)
and progenitor cells that persist in the subependymal zone into
adulthood (Gritti et al., 1996). In addition, FGF2 regulates the fate
of VZ progenitor cells in vitro, with low concentrations
favoring neuronal differentiation, higher threshold concentrations
favoring oligodendroglial differentiation, and additional environmental
factors required for astrocytic differentiation (Qian et al., 1997). In
contrast to the proliferative effect of FGF2 on early VZ progenitor
cells, EGF is a potent mitogen for later multipotent progenitor cells
from the embryonic and adult SVZ (Reynolds and Weiss, 1992; Reynolds et
al., 1992).
Increasing evidence suggests that the fate of neural progenitors is
regulated by members of the bone morphogenetic protein (BMP) subclass
of the TGF superfamily. The BMPs have multiple functions in
embryogenesis and in neural development. In addition to their complex
roles in the development of neural crest and the peripheral nervous
system (Fann and Patterson, 1994; Liem et al., 1995; Lien et al., 1995;
Sasai et al., 1995; Wilson and Hemmati-Brivanlou, 1995; Mujtaba et al.,
1998), they promote the differentiation of neuronal precursors in the
spinal cord (Kalyani et al., 1998) and cortex (Li et al., 1998) and
promote astroglial lineage commitment by forebrain SVZ progenitor cells
(Gross et al., 1996). In certain contexts, the BMPs also induce
apoptosis of neural cells (Graham et al., 1994; Glozak and Rogers,
1996; Song et al., 1998), including early telencephalic neuroectoderm (Furuta et al., 1997). BMPs and their receptors are abundantly expressed in the brain from early embryogenesis throughout adult life
(Furuta et al., 1997; Mehler et al., 1997; Zhang et al., 1998). BMP
actions are regulated in vivo by proteins such as noggin, chordin, and follistatin, which antagonize BMP signaling by directly binding BMPs and blocking ligand activity (for review, see Cho and
Blitz, 1998). In addition, in many contexts, the effects of the BMPs
are opposed by other growth factors, such as EGF and FGFs, which are
thought to interfere with the intracellular BMP signaling pathway
(Bernier and Goltzman, 1992; Niswander and Martin, 1993; Ganan et al.,
1996; Kretzschmar et al., 1997; Neubuser et al., 1997).
These observations collectively suggest that the BMPs play a
fundamental role in many aspects of embryogenesis of the nervous system. The precise actions of this family of proteins at different stages of ontogeny, in particular the relationship between the promotion of apoptosis and differentiation, however, remain unclear. Our results demonstrate that the BMPs have complex effects on the
survival and differentiation of developing cortical cells. At embryonic
day 13 (E13), at approximately the onset of cortical neurogenesis, high
concentrations of the BMPs (10-100 ng/ml) promote apoptotic death and
inhibit the proliferation of cultured VZ progenitor cells, resulting in
a decrease in the generation of both neurons and glia. In identical
culture conditions at E16, near the midpoint of neurogenesis, moderate
concentrations of the BMPs (1-10 ng/ml) promote neuronal
differentiation, and to a lesser extent astrocytic differentiation,
while completely preventing the development of oligodendroglia. These
observations demonstrate that BMP effects on CNS cells are
context-specific and change during ontogeny.
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MATERIALS AND METHODS |
Cell cultures. Timed pregnant Sprague Dawley rats
were killed by CO2 narcosis at either
embryonic day 13 (E13) or E16 (counting the morning of the vaginal plug
as day 1), and embryos were aseptically removed and placed in Puck's
saline G. Dorsolateral cerebral cortex was dissected and enzymatically
dissociated with trypsin 0.05% for 5 (E13) or 10 (E16) min, mixed with
an equal volume of Neurobasal medium (Life Technologies, Gaithersburg,
MD) supplemented with 10% fetal bovine serum, centrifuged at
700 rpm for 5 min, placed in serum-free medium (SFM), triturated, and
then passed through a 20 µM nytex filter. After
counting viability by trypan blue exclusion, cells were plated in a
volume of 10 µl onto poly-D-lysine (PDL)-coated (20 µg/ml) Terasaki plates in SFM at low (~10
cells per well) or moderate (250-300 cells per well) density. For some moderate density experiments, cells were plated in 24-well plates at
2 × 104 cells per well. SFM
consisted of Neurobasal medium supplemented with N2 and B27 (Life
Technologies), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were maintained in a humidified incubator at 37° C
with 5% CO2. An additional 5 µl of SFM was
added to each well at 4-5 days in vitro (div). BMP2
and FGF2 were added only once at the time of plating, whereas noggin
was readded with SFM at 4-5 div. For clonal analysis, three to four
cells were plated per Terasaki well or 300-400 cells were plated onto
PDL-coated 35 mm plates with gridded bottoms (Nunc, Naperville, IL),
and individual viable cells were marked 1-2 hr later. Cultures were
then maintained as described above for low-density cultures, with the
exception that fibronectin (1 µg/ml) was added to the SFM at the time
of plating.
Growth factors. Recombinant human BMPs 2 and 4 were provided
by A. Celeste and J. Wozney of Genetics Institute (Cambridge, MA).
Recombinant human noggin was provided by A. Economides and G. Yancopoulos of Regeneron Pharmaceuticals (Tarrytown, NY). FGF2 was
purchased from Collaborative Biomedical (Bedford, MA).
Immunocytochemistry. At the designated time, cells were
fixed with cold methanol for 10 min and washed twice with PBS.
For oligodendroglial (O4) staining, cells were incubated with O4
supernatant for 20 min at room temperature and rinsed with PBS before
fixation. Primary antibodies diluted in PBS containing 5%
heat-inactivated goat serum were applied for 2-4 hr. After three
washes with PBS, species- and isotype-specific fluoroscein and/or
rhodamine-labeled secondary antibodies (Southern Biotechnology,
Alabaster, AL) diluted 1:100 and containing 1 µg/ml bisbenzimide were
applied at room temperature for 1 hr. Cell staining was visualized and
photographed with an Olympus Opticals (Tokyo, Japan) fluorescent
microscope. Primary antibodies (mouse monoclonals) used were anti-
tubulin III (1:100-1:400, depending on lot; Sigma, St. Louis, MO),
anti-glial fibrillary acidic protein (GFAP) (1:400; Sigma),
anti-nestin (1:1000; PharMingen, San Diego, CA), and O4 (supernatant
diluted 1:2-5 from the mouse hybridoma cell line; the gift of S. Pfeiffer, University of Connecticut). Some cultures were triple-labeled
using a combination of fluorescent and biotin-avidin-peroxidase
(Vector ABC kit; Vector Laboratories, Burlingame, CA) stains.
Immunohistochemistry. The brains of E16 rats and E15 and
newborn mice were fixed by immersion in 0.5% zinc acetate, 0.5% zinc chloride, and 0.05% calcium acetate in 50 mM
Tris, pH 7.2 for 1-2 d (Beckstead, 1994). After cryoprotection in 30%
sucrose in 50 mM Tris, tissue was frozen in
isopentane, and 12 µm cryosections were placed onto Superfrost
Plus slides (Fisher Scientific, Houston, TX) and air-dried overnight.
Sections were treated with 0.3%
H2O2 in methanol for 20 min
to permeabilize membranes and quench endogenous peroxidase activity.
Primary antibodies were applied for 2-4 hr, followed by fluorescent
antibody (as above) or biotinylated secondary antibodies, ABC reagent,
and the VIP substrate kit (Vector Laboratories) following the
manufacturer's protocol. Primary antibodies used were a mouse
monoclonal anti-BMP2 diluted 1:4000 (the gift of Genetics Institute),
an affinity-purified goat polyclonal anti-BMP2 diluted 1:100 (Research
Diagnostics, Flanders, NJ), and two different affinity purified rat
monoclonal anti-noggin antibodies (RP57-16 and RP57-21; gift of
Regeneron Pharmaceuticals) diluted 1:500 and 1:100, respectively.
Specificity of staining was demonstrated by the following criteria: (1)
the absence of any detectable staining with several irrelevant
monoclonal antibodies, (2) a correlation between intensity of
immunoreactivity and the documented level of transcript expression in
the same tissue by Northern blot (Valenzuela et al., 1995) or nuclease
protection assay (Gross et al., 1996), and (3) an identical pattern of
immunoreactivity using two different antibodies.
Quantification. The number of viable cells per Terasaki well
was counted at sequential time points before fixation in at least 30 wells for low-density and 10 wells for moderate-density cultures. Morphological criteria for determining viability were the presence of
an intact cell membrane and processes and the absence of significant cell shrinkage and/or formation of membrane-bound bodies under 20×
phase microscopy. Nonviable cells consistently exhibited shrinkage of
the soma to <50% of the size of average cells in the same well, retraction and/or disintegration of processes, and nuclear pyknosis or
the formation of membrane-bound (apoptotic) bodies containing nuclear
fragments. With close serial inspection of low-density cultures,
remnants of virtually all nonviable cells remained visible for several
days, allowing for a precise assessment of proliferation and viability.
In multiple experiments, a subset of wells were analyzed by trypan blue
exclusion and/or bisbenzimide nuclear staining within 45 min of
morphological assessment of viability, confirming the accuracy of the
morphological criteria to within 2-3%. Similar results were seen in
at least three separate culture experiments. Results are expressed as
the mean ± SEM, with significance determined by the t test.
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RESULTS |
To define the effects of the BMPs on cortical progenitor cell
survival, proliferation, and differentiation, cortical cells from E13
and E16 rats were treated with the BMPs in several culture conditions:
one that favors proliferation and subsequent differentiation of both
neurons and glia (FGF2 10 ng/ml), one that favors more limited
proliferation and subsequent neuronal differentiation (FGF2 0.1 ng/ml),
and one that favors early neuronal differentiation (no FGF2) (Qian et
al., 1997). Cultures grown at both moderate and low densities were also
compared to help determine whether BMP effects are influenced by
autocrine-paracrine signaling.
BMPs are negative regulators of cortical VZ cell number
The first set of experiments examined rat E13 cortical cells
plated at ~10 cells per Terasaki well in serum-free medium
supplemented with FGF2 10 ng/ml. This concentration of FGF2 promotes
the proliferation of multipotent VZ progenitor cells plated at low
density and the generation of both neurons and glia (Qian et al.,
1997). At the time of plating, 92 ± 6% of E13 cortical cells
expressed nestin, a marker for undifferentiated neuroepithelial cells.
No cells expressed detectable astrocytic (GFAP) or O4 markers, but
16 ± 5% expressed tubulin III, a neuronal marker (the small
population of dual-immunoreactive cells presumably represents a
transient developmental stage at the onset of neuronal
differentiation). In the absence of FGF2, all cells died within 3 d. In the presence of FGF2, there was extensive proliferation of
nestin-immunoreactive cells and a significant increase in the number of
tubulin III-immunoreactive neurons over 8 div (Figs.
1, 2). When
E13 cortical cells were grown with FGF2 plus increasing concentrations
of BMP2, the number of cells generated was markedly decreased (Figs. 1,
2). The trend toward decreased cell numbers after BMP2 treatment was
apparent within 2 div and reached statistical significance at 3 div.
Serial cell counts of viable and nonviable cells revealed that BMP
treatment increased the percentage of dying cells and decreased the
rate of proliferation. Cell viability was quantified by morphological analysis and confirmed by trypan blue exclusion (see Materials and
Methods). At 5 div, viability was 80 ± 7% with FGF2, 62 ± 6% with FGF2 plus BMP2 10 ng/ml (p = 0.021),
and 42 ± 7% with FGF2 plus BMP2 100 ng/ml
(p = 0.0048). Dying cells typically displayed shrinkage of the soma with the formation of membrane-bound bodies under
phase microscopy and pyknosis and/or fragmentation of the nucleus with
bisbenzimide staining (Fig. 1), the morphological hallmarks of
apoptosis (Kerr et al., 1995). Dying cells included both neurons
(tubulin III-immunoreactive) and neuroepithelial cells
(nestin-immunoreactive), without apparent selectivity.
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Figure 1.
Effects of BMP2 on the growth of cortical VZ cells
plated at low density with FGF2 10 ng/ml. Photomicrographs of rat E13
cortical cells grown with FGF2 alone (A,
D, G, J), FGF2 plus
BMP2 10 ng/ml (B, E, H,
K), or FGF2 plus BMP2 100 ng/ml
(C, F, I,
L). A-C, Phase-contrast images at 5 div.
D-F, Bisbenzimide staining at 8 div.
G-I, tubulin III staining at 8 div.
J-L, Nestin staining at 8 div. The same photographic
field is shown for each condition in D,
G, J; E, H,
K; and F, I,
L at 8 div. Arrows indicate examples of
dying cells with apoptotic bodies (A-C), cells
expressing tubulin III-immunoreactivity
(G-I), and nestin immunoreactivity
(J-L). In each condition, an occasional cell
coexpressed both tubulin III and nestin (for example, the
top cell indicated with an arrow in
I and L). Scale bar: A-C,
8 µM; D-L, 12 µM.
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Figure 2.
Effects of BMP2 on the growth of cortical VZ
cells. The number of total viable cells was quantitated at the
indicated time points. A, Effects of BMP2 10-100 ng/ml
on E13 cells grown at low density with FGF2 10 ng/ml for 8 div.
B, Effects of BMP2 10 ng/ml on cells grown at moderate
density with FGF2 0.1-10 ng/ml for 9 div. C, Effects of
BMP2 0.1-100 ng/ml on E13 cortical cells grown at moderate density
without FGF2 for 5 div. *p < 0.05;
**p < 0.01.
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Similar results were obtained when E13 cortical cells were plated at
moderate density (Fig. 2). Trypan blue viability assays at sequential
time points from 3 to 8 div demonstrated that BMP2 cotreatment
increased the percentage of dead cells (data not shown). In addition,
proliferation assays of moderate-density cultures demonstrated that
77 ± 2% of cells treated with FGF2-incorporated bromodeoxyuridine at 3 div compared with only 59 ± 3% of
cells cotreated with BMP2 10 ng/ml (p = 0.00048). A similar reduction in the percentage of cells expressing
proliferating cell nuclear antigen (PCNA) was present at 6 div (data
not shown). Thus, in both low-density and high-density cultures, the
net decrease in cell number resulted from both increased cell death and
decreased proliferation. At 8 div, BMP treatment decreased the numbers
of both neurons ( tubulin III-immunoreactive) and undifferentiated neuroepithelial cells (nestin-immunoreactive), without changing their
relative proportion (no differentiated glial cells were detected at
this time). At 12 div, however, FGF2-treated cultures contained 20 ± 4% oligodendrocytes (O4-immunoreactive) and 42 ± 4%
astrocytes (GFAP-immunoreactive). Differentiated glia were absent in
cultures cotreated with BMP2 10 ng/ml. Thus, BMP treatment inhibited
the net production of both neurons and glia. BMP4 had similar effects,
except that its potency was somewhat greater than BMP2 at equivalent
doses (data not shown).
To examine the effects of the BMPs in conditions that favor more
restricted neuronal development, additional experiments were performed
at moderate density in the absence or presence of lower doses of FGF2
(0.1 ng/ml). E13 cortical cells cultured at this dose of FGF2 exhibited
less proliferation than cells grown in 10 ng/ml FGF2, and 81 ± 8% differentiated into neurons by 6 div. BMP2 (10 ng/ml) had two
effects. First, BMP2 markedly decreased the number of cells generated
(Fig. 2B). As with cultures grown in the higher dose
of FGF2 (10 ng/ml), the reduction in cell number reflected increased
cell death and decreased proliferation, and again, the proportion of
neurons to undifferentiated cells was not significantly altered (data
not shown).
E13 cortical cells cultured in the absence of FGF2 displayed limited
proliferation, with 90 ± 4% of cells differentiating into
neurons within 4 div. Increasing doses of BMP2 dramatically decreased
the viability of neurons (Fig. 2C). Thus, the BMPs
negatively regulated E13 cortical cell number in conditions that both
favored proliferation and fostered differentiation. Higher
concentrations of BMP2 were required to significantly reduce cell
numbers in the absence of FGF2, suggesting that the proliferative
effect of FGF2 may sensitize E13 cortical cells to the death-inductive and anti-mitotic effects of the BMPs. In all of these conditions, those
neurons that survived in the presence of the BMPs typically displayed
more extensive neuritic outgrowth than control neurons (data not shown)
(Li et al., 1998).
Moderate doses of BMPs promote neuronal differentiation and inhibit
oligodendroglial differentiation of E16 cortical cells
Cultures of E16 cortical cells grown in serum-free medium plus
FGF2 displayed several differences from E13 cells. As expected, a
higher percentage of cells at the time of plating already expressed the
neuronal marker tubulin III (35 ± 4% for E16; 16 ± 5%
for E13), a lower percentage of cells expressed the neuroepithelial marker nestin (69 ± 7% for E16; 92 ± 6% for E13), and
accordingly, fewer cells initially proliferated in response to FGF2. In
addition, glial cells differentiated more quickly, reaching significant levels within 8 div (Figs. 3,
4B). In cultures plated
at low density, many oligodendrocytes had differentiated by 8 div,
whereas astrocytes rarely differentiated, even after 12 div. At
moderate density, both oligodendrocytes and astrocytes were present by
8 div. The effects of BMP treatment on E16 cortical cells also
significantly differed from E13 cells. First, concentrations of BMP2 in
the 1-10 ng/ml range did not significantly alter total cell number in
low-density cultures (Fig. 4A). However, the
phenotype of the cells that developed was significantly altered. By 8 div, BMP treatment dramatically increased the percentage of neurons
that developed and virtually abolished the generation of
oligodendrocytes (Figs. 3, 4B). These effects
occurred without altering the percentage of undifferentiated cells
(Fig. 4B). Furthermore, there was no significant
effect on viability or proliferation based on serial cell counts (Fig.
4A) and PCNA expression. At 3 div, PCNA was expressed
by 84 ± 4% of FGF-treated cells and by 82 ± 5% of FGF2 plus BMP2-treated cells. PCNA was coexpressed by
nestin-immunoreactive cells but never by tubulin III-immunoreactive
cells; this suggests that the increase in neurons seen with BMP
treatment reflected enhanced neuronal differentiation of
neuroepithelial progenitor cells rather than proliferation of cells
already expressing the neuronal phenotype.
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Figure 3.
Effects of BMP2 on E16 cortical cells grown at low
density with FGF2 10 ng/ml. Photomicrographs of cells grown with FGF2
alone (A, C, E) or FGF2
plus BMP2 10 ng/ml (B, D,
F) for 8 div. A, B,
Bisbenzimide staining. C, D, tubulin
III staining. E, F, O4 staining. The same
photographic field is shown for each condition. Scale bar, 12 µM.
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Figure 4.
Effects of BMP2 on E16 cortical cells. The number
of total viable cells and cells expressing neuronal ( tubulin III),
oligodendroglial (O4), and neuroepithelial (nestin) markers was
quantitated at the indicated time points. A, Effects of
BMP2 on E16 cells grown at low density with FGF2 10 ng/ml.
B, Effects of BMP2 on the differentiation of E16
cortical grown at low density with FGF2 10 ng/ml for 8 div.
C, Effects of BMP2 0.1-100 ng/ml on E16 cortical cells
grown at moderate density without FGF2. *p < 0.05;
**p < 0.01.
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To confirm these results, a clonal analysis was performed. Cells were
plated at clonal density (see Materials and Methods), and the fate of
individual cells was then followed for 8 d (Table 1). The rate of survival of single nondividing cells and
cells that generated small (two to four cells) and large (more than four cells) clones was similar between FGF2 alone and FGF2 plus BMP2.
Likewise, the phenotype of single cells and small clones was similar
between the two groups. The large clones, however, differed in several
respects. As predicted based on the low-density results above, large
FGF2 plus BMP2-treated clones were more likely to contain neurons, and
those that did contained a higher percentage of neurons than large
FGF2-treated clones. Again, BMP2 completely inhibited the development
of oligodendroglia. BMP2-treated clones tended to be smaller,
reflecting decreased proliferation and slightly increased death. These
findings suggest that the BMPs operate predominantly within multipotent
clones to increase neuronal and inhibit oligodendroglial development
and that they are not survival or proliferative factors for
lineage-restricted neuronal precursors.
High BMP concentrations reduce E16 cortical cell number in both
proliferative and differentiating conditions
To define the effects of higher concentrations of BMP, E16
cortical cells were treated with 100 ng/ml BMP2 in either the presence of 10 ng/ml FGF2 (to maximally stimulate proliferation) or the absence
of FGF2 (to promote neuronal differentiation). This dose of BMP2
dramatically reduced the number of E16 cortical cells in low-density
cultures in the presence of FGF2 (Fig. 4A). The number of viable cells increased between 0 and 8 div in every well
containing FGF2 alone, whereas cell numbers actually decreased in 30%
of wells grown with FGF2 plus BMP2 100 ng/ml. Assessment of viability
at 4 div (exclusion of trypan blue) revealed that 83 ± 4% of
cells were viable in wells containing FGF2, whereas only 42 ± 4%
were viable in the presence of FGF2 plus BMP2 (p < 0.0001). As was seen in E13 cultures, BMP treatment also reduced proliferation based on serial cell counts (Fig. 4A)
and PCNA expression (at 3 div, FGF2, 84 ± 4%; FGF2 plus BMP2,
62 ± 5%; p = 0.018). Moreover, the percentage of
neurons was increased, and no oligodendrocytes were present after BMP2
cotreatment (data not shown). When E16 cortical cells were grown at
moderate density in the absence of FGF2, there was limited
proliferation, and, by 4 div, almost all surviving cells had
differentiated into neurons. In this context, BMP2 (100 ng/ml) induced
the death of immature neurons (Fig. 4C). Thus, as was seen
with E13 cells, high concentrations of BMP2 markedly decreased cell
number in both proliferative and differentiating conditions. Again,
surviving neurons still displayed enhanced neuritic outgrowth (data not shown).
BMP effects on cell number and neuronal differentiation are
temporally dissociable from suppressive effects on oligodendroglial
differentiation
The opposing effects of the BMPs on neuronal and oligodendroglial
development could reflect several possible mechanisms. First, the BMPs
could influence the fate of a multipotent or bipotent neuronal-oligodendroglial progenitor cell (Williams and Price, 1995),
directly promoting neuronal differentiation at the expense of
oligodendroglial differentiation. Alternatively, the BMPs could have
selective effects on two separate subpopulations of cells: enhanced
differentiation of neuronal precursors and decreased survival and/or
differentiation of oligodendroglial precursors. To help distinguish
between these two possibilities, we performed an additional experiment
in which cells were grown at low density in FGF2 alone for the first 4 div and then in FGF plus BMP2 (10 and 100 ng/ml) for the subsequent 4 div. Delaying the addition of BMP2 altered its effects on both cell
number and phenotype (Fig. 5). Total cell
numbers were unaffected rather than dramatically decreased, with
delayed addition of BMP2 100 ng/ml. Neuronal differentiation was
somewhat decreased rather than increased by delayed addition of BMP2 at
either concentration. Oligodendroglial differentiation, however, was
still inhibited after delayed addition of BMP2, and a greater
percentage of undifferentiated cells were present (Fig. 5). Thus, the
effects of BMP2 on the generation and differentiation of cortical cells
changed profoundly over several days, and there was a temporal
dissociation between effects of BMP2 on cell number and neuronal
differentiation (which were lost after delayed addition) versus effects
on oligodendroglial differentiation (which persisted). Furthermore,
delaying exposure to the BMPs also significantly increased astrocytic
differentiation (see below).
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Figure 5.
Effects of delaying the addition of BMP2 on
E16 cortical cell differentiation. Cells were grown at low density with
FGF2 alone for the first 4 div, and then BMP2 was added at 10 or 100 ng/ml. The number of cells expressing neuronal ( tubulin III),
oligodendroglial (O4), astrocytic (GFAP), and neuroepithelial (nestin)
markers was quantitated at 8 div. *p < 0.05;
**p < 0.01.
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BMPs promote astrocytic differentiation
BMP treatment promoted astrocytic differentiation of both
EGF-generated SVZ progenitor cells and O2A progenitor cells
(Gross et al., 1996; Mabie et al., 1997). In the present study,
however, few astrocytes were found in low-density E16 cultures, even in the presence of BMP treatment. This raised the question of whether these cells were intrinsically limited in their potential for astrocytic differentiation or, alternatively, whether the combination of FGF2 plus BMP2 was insufficient to promote astrocytic
differentiation at this developmental stage. To examine astrocytic
development more fully, two additional experiments were performed.
First, as mentioned above, E16 cortical cells were plated at low
density in FGF2 alone for 4 div before BMP2 treatment. In addition to the effects described above, delaying the addition of BMP2
significantly increased the number of astrocytes (Fig. 5), suggesting
that the responsiveness of early cortical cells to the
astrocyte-inductive effects of the BMPs increases as they develop.
Second, we examined the effects of FGF2 plus BMP2 cotreatment on E16
cortical cells plated at moderate density. In contrast to low-density
cultures, a moderate number of astrocytes developed in moderate-density cultures treated with FGF2 alone for 8 div. In these cultures, BMP
cotreatment promoted astrocytic differentiation; astrocytes differentiated more rapidly and constituted a higher percentage of
total cells. FGF2-treated cultures contained 8 ± 2% astrocytes at 5 div and 35 ± 3% astrocytes at 8 div. BMP2 (10 ng/ml)
cotreated cultures contained 19 ± 2% astrocytes at 5 div
(p = 0.04) and 50 ± 4% astrocytes at 8 div (p = 0.01). Consistent with low-density cultures, the percentages of neurons and oligodendrocytes were increased and decreased, respectively (data not shown). Thus, within a
permissive environment, the BMPs promoted astrocytic differentiation of
E16 cells.
Noggin, a BMP antagonist, promotes oligodendroglial differentiation
and inhibits neuronal differentiation
In many developmental contexts, BMP signaling is inhibited by
endogenous antagonists that limit the availability of ligand to
receptors. At least one of these antagonists, noggin, is expressed in
the developing cortex (Valenzuela et al., 1995), raising the possibility that intrinsic BMP signaling within the cortex is regulated
in this fashion. E16 cortical cells were therefore cultured at low
density with FGF2 in the absence or presence of noggin (100 ng/ml).
This concentration was chosen based on its ability to block the effect
of 10 ng/ml of BMP2 (data not shown). Noggin cotreatment significantly
increased the number of oligodendrocytes without altering the total
number of cells (Figs. 6,
7). Noggin also consistently decreased
the percentage of neurons that differentiated, although this effect did
not reach statistical significance (Figs. 6, 7). This suggests that
endogenously produced BMPs may influence the development of cultured
cortical cells by inhibiting oligodendroglial differentiation and
promoting neuronal differentiation. Furthermore, cell survival and
differentiation in the developing cortex may be coordinately regulated
by BMP antagonists, as well as by the BMPs themselves.
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Figure 6.
Effects of noggin on E16 cortical cells grown at
low density with FGF2 10 ng/ml. Photomicrographs of cells grown with
FGF2 alone (A, C) or FGF2 plus noggin 100 ng/ml (B, D) for 8 div. A,
B, tubulin III staining. C,
D, O4 staining. Scale bar, 15 µM.
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Figure 7.
Effects of noggin on E16 cortical cells grown at
low density with FGF2 10 ng/ml. The number of cells expressing neuronal
( tubulin III), oligodendroglial (O4), and neuroepithelial (nestin)
markers was quantitated at 8 div. *p < 0.05;
**p < 0.01.
|
|
BMP2 and noggin are expressed in the developing cortex
Although expression of transcripts for BMP ligands and receptors
within the forebrain has been well documented (Furuta et al., 1997;
Mehler et al., 1997; Zhang et al., 1998), knowledge of ligand
localization is limited. Based on recent immunohistochemical evidence
demonstrating BMP2 and/or BMP4 protein along the cortical ventricular
lining during the period of neurogenesis (Li et al., 1998) and the
results of nuclease protection assays (unpublished observations) and
in situ hybridizations (Furuta et al., 1997), it seems
likely that protein localization is more widespread than the level of
transcript detectable by in situ hybridization. In the case
of BMP4, one possible explanation for this is the presence of BMP4
transcripts in the presumptive choroid plexus, whereby protein released
into the CSF could easily reach cells lining the ventricular
system. Many BMPs are also highly expressed in the placenta and in
endothelial cells and thus at stages of CNS development before the
formation of a strict blood-brain barrier, BMPs may enter the CNS from
the circulation.
To examine the distribution of BMP2 protein in the developing cortex,
embryonic and neonatal brains were analyzed by immunohistochemistry with antibodies to BMP2. In E16 rat brain, BMP2 protein was expressed abundantly throughout the cortex, from the ventricular lining to the
pial surface (Fig. 8A).
In the neonatal cortex, BMP2 staining was again prominent in the
cortical plate and ependymal/subependymal region but was absent in the
developing subcortical white matter/corpus callosum (Fig.
8B). This pattern of BMP2 protein distribution coincides with the pattern of BMP receptor subunit transcripts at these
developmental stages (Zhang et al., 1998).
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Figure 8.
BMP2 and noggin expression in the developing
cortex. Photomicrographs of E16 rat (A) and
neonatal (B, C) murine cortex stained
with a monoclonal antibody to BMP2 (A, B)
and a monoclonal antibody to noggin (C). The
arrows in A indicate the pial (top) and
ventricular (bottom) surfaces. For neonatal coronal
sections (B, C), the
arrows in C indicate the position of
(from top to bottom) the pial surface,
the border between cortical gray matter and developing subcortical
white matter, the border between subcortical white matter and
subventricular zone, and the lateral ventricle.
|
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Because the BMP antagonist noggin is also known to be expressed within
the developing cortex (Valenzuela et al., 1995), we also examined its
distribution. In agreement with the low level of noggin transcript in
the early cortex (Valenzuela et al., 1995), noggin protein was
undetectable by immunohistochemistry in E15 mouse cortex (data not
shown). In neonatal mice, noggin protein was abundant in the developing
subcortical white matter/corpus callosum and was much less apparent
throughout the remainder of the cortex (Fig. 8C).
This suggests that, during the major period of neurogenesis and
gliogenesis, BMP2 signaling is most active near the ventricular lining
and in the cortical plate. In contrast, in the intermediate zone and
developing subcortical white matter, BMP2 signaling is less active
because of a decrease in the amount of ligand and an increase in
the amount of antagonist. Thus, interactions between BMP2 and noggin
may regulate cortical cell development by defining spatial
gradients of BMP signaling, similar to what has been postulated in
earlier patterning events (Sasai et al., 1995; Wilson and
Hemmati-Brivanlou, 1995; Hogan, 1996).
| |
DISCUSSION |
The BMPs appear to play multiple roles in the regulation of
cortical cell fate. Effects of BMP signaling vary depending on the
timing of exposure, the dose, and the presence of other environmental signals. Early in cortical development (E13), the predominant effects
of BMP signaling are the induction of cell death and the inhibition of
proliferation. At E16, moderate doses of the BMPs (1-10 ng/ml) promote
neuronal differentiation. Under permissive conditions, and increasingly
with time, they induce astrocytic differentiation of older progenitors.
Oligodendroglial differentiation is inhibited at all stages. Although
these effects suggest complex regulatory mechanisms underlying BMP
signaling, similar kinds of sequential actions have been reported for
the BMPs in earlier neural development and in other organ systems (Fann
and Patterson, 1994; Liem et al., 1995; Lien et al., 1995; Sasai et
al., 1995; Wilson and Hemmati-Brivanlou, 1995; Reissmann et al., 1996;
Shah et al., 1996; Jernvall et al., 1998; Kalyani et al., 1998; Mujtaba et al., 1998). Our data are consistent with previous reports analyzing the effects of the BMPs on cortical cells (Furuta et al., 1997; Li et
al., 1998). By demonstrating how the concentrations of BMPs and
mitogenic growth factors (i.e., FGF2) influence the choice between
death and differentiation, our study clarifies the apparent contradictory role of the BMPs in promoting cortical apoptosis and
neuronal differentiation. Our use of low-density and clonal analysis to
follow cell fate over longer culture periods has enabled us to define
the effect of BMP signaling on multipotent progenitor cells and to
clarify the role of BMP signaling on glial development. Our
observations suggest that BMP signaling may be important at multiple
stages of neural development and that the biological responses of
cortical progenitor cells to the BMPs differ depending on the
prevailing developmental context.
BMP induction of cortical cell death
Although estimates of the proportion of cells undergoing apoptosis
during corticogenesis vary widely depending on the method of detection
(Finlay and Slattery, 1983; Ferrer et al., 1992; Wood et al.,
1992; Blaschke et al., 1996; Takahashi et al., 1996; Furuta et al.,
1997; Price et al., 1997), cell death has been increasingly recognized
as an important mechanism for the regulation of cell number and
phenotype within the developing cortex. In our studies, the cells dying
in response to BMP treatment almost always displayed morphological
hallmarks of apoptotic cell death, including cell shrinkage, pyknosis,
and the formation of membrane-bound nuclear fragments (apoptotic
bodies) (Kerr et al., 1995). Although we did not use any biochemical
assays for apoptosis, the BMPs have been implicated in apoptosis of
other neural (Graham et al., 1994; Glozak and Rogers, 1996; Furuta et
al., 1997; Song et al., 1998), as well as mesenchymal (Niswander and
Martin, 1993; Zou et al., 1997; Jernvall et al., 1998), cell types.
BMP-induced death of cortical progenitor cells was accompanied by an
anti-mitotic effect, suggesting that death might result, in part, from
the incompatibility of mitotic (FGF2) and anti-mitotic (BMP) signaling (Freeman et al., 1994; Park et al., 1996, 1998). This hypothesis is
supported by the increased susceptibility to BMP-induced death after
cotreatment with the mitogen FGF2 and the observation that E13 cortical
cells, which proliferate more rapidly than E16 cells, are more
sensitive to the death-inductive effect of the BMPs than E16 cells. The
increased sensitivity of E13 cells to exogenous BMPs may also reflect a
higher level of endogenous BMP signaling at this developmental stage.
High concentrations of BMP2 also induced death of postmitotic neurons
in the absence of FGF2, indicating that BMP-induced death may occur by
mechanisms other than conflicting signaling influences on the cell
cycle. Another hypothesis is that the BMPs promote death by inducing
premature or inappropriate dependence on other survival factors. For
example, treatment of immortalized sympathoadrenal progenitor with BMP2
induces their dependence on exogenous growth factors for survival, and
BMP-treated cells undergo apoptosis in the absence of such trophic
support (Song et al., 1998). Presumably, the BMPs initiate a cell death program unless it is suppressed by exogenous growth factors.
BMP regulation of cortical cell differentiation
The effects of the BMPs on neural progenitor cell differentiation
in vitro are strikingly dependent on the developmental stage of the cells and the culture conditions. At concentrations lower than
those promoting cell death, we found that BMP treatment promoted neuronal differentiation of E16, but not E13, cortical progenitor cells. Our clonal analysis demonstrates that exposure to the BMPs dramatically increases the frequency of clones containing neurons, as
well as the proportion of neurons that differentiate within neuron-containing clones, while simultaneously eliminating the generation of oligodendroglia. These findings suggest that the BMPs
influence the development of cortical lineages by an instructive mechanism on multipotent cells (increasing the probability of neuronal
differentiation and preventing oligodendroglial development within
single multipotent clones). The presence of these differentiating effects in microwells containing less than four cells and the high
levels of BMP receptors in the cortical ventricular zone (Zhang et al.,
1998) suggest that these are direct effects of BMP signaling. The
absence of a neuronogenic effect on E16 cells when BMP exposure is
delayed for 4 div demonstrates that this effect of exogenous BMP occurs
only within a relatively brief developmental window.
The effects of BMPs on E16 cortical progenitors reported here differ
dramatically from those observed with cultured EGF-generated SVZ
progenitor cells in which BMP treatment promotes astrocytic differentiation at the expense of neurons and oligodendroglia (Gross et
al., 1996). Similarly, BMP treatment of FGF-generated progenitor cells
from the perinatal SVZ promotes astrocytic differentiation, inhibits
oligodendroglial differentiation, and has no effect on neuronal
differentiation (our unpublished observations). Thus, there are
differences in progenitor cells from different developmental stages
irrespective of the mitogen used to support the cells in culture.
Although few astrocytes were found in low-density E16 cultures, even in
the presence of BMP2, treatment with BMP2 significantly increased
astrocytic differentiation in higher density cultures, suggesting the
need for a cofactor to generate astrocytes at this early developmental
stage. However, delaying the addition of BMP2 to E16 cortical cells
significantly increased the number of astrocytes, even in low-density
cultures, indicating that the responsiveness of early cortical cells to
the astrocyte-inductive effects of the BMPs increases as they develop.
Previous studies have demonstrated that CNTF treatment induces
differentiation of embryonic progenitors into GFAP-immunoreactive cells
(Johe et al., 1996). Addition of BMP2 along with CNTF further increased
the percentage of GFAP-immunoreactive cells (data not shown),
indicating that the effects of BMP2 are additive to those of CNTF at
E16. BMP2 treatment also induced a more stellate morphology
characteristic of astrocytes. Thus, at earlier embryonic stages (e.g.,
at approximately E16), the BMPs may be involved in enhancing astrocytic
differentiation after induction of the phenotype by cofactors such as
CNTF. At later stages, BMPs are sufficient to both induce and enhance
astrocytic differentiation (Gross et al., 1996; Mabie et al., 1997),
even in the absence of CNTF signaling (Koblar et al., 1998). We
hypothesize, therefore, that postnatal astrocyte differentiation may
primarily reflect BMP signaling. This hypothesis is consistent with
previous suggestions that there may be more than one developmental
pathway for astrocytic differentiation (Cameron and Rakic, 1991; Koblar et al., 1998; Rajan and McKay, 1998).
A consistent finding in all of our work with BMPs and CNS progenitor
cells is the inhibition of oligodendroglial development. This finding
is present with both embryonic and postnatal cultures derived from
cortex and other regions and occurs independent of whether cells are
initially grown in serum or mitogenic growth factors, such as FGF2 or
EGF. In light of our previous studies of EGF-generated SVZ cells and
O2A cells (Gross et al., 1996; Mabie et al., 1997), the results
reported here suggest that the mechanisms underlying this suppressive
effect are complex. At early developmental stages (e.g., E13), the
predominant mechanism of this suppressive effect is most likely the
death of a precursor pool. At later developmental stages (e.g.,
perinatal) the predominant mechanism is the induction of astroglial
differentiation of a bipotent glial progenitor cell at the expense of
oligodendroglial differentiation (Mabie et al., 1997). At an
intermediate stage (e.g., E16), the data presented here suggests that
oligodendroglial inhibition occurs by a block in the differentiation
pathway, resulting in the acquisition of the neuronal phenotype and/or
the retention of an undifferentiated phenotype.
These results have interesting implications for the study of
oligodendrogliogenesis. Several recent studies have demonstrated that,
although progenitors competent to differentiate into oligodendrocytes have a widespread distribution in the early CNS, in vivo the
earliest oligodendroglial progenitors arise in relatively discrete
regions along the ventral neural axis (Cameron-Curry and Le Douarin,
1995; Hardy and Friedrich, 1996; Orentas and Miller,
1996). Over time, oligodendrocytes become more widespread by
either migration and/or a process of progressive inductive signaling.
In the forebrain, the progeny of bipotent glial progenitors within the
SVZ adopt an astrocytic or oligodendroglial fate depending on their
postmigratory environment (Levison and Goldman, 1993). We propose that
the anatomic restriction of oligodendroglial development is regulated
in part by the level of BMP signaling. Support for this hypothesis
comes from the loss of ventral neural tube cell types in the noggin knock-out mouse (McMahon et al., 1998). Together, these results suggest
that oligodendrocyte development requires inhibition of BMP signaling.
In summary, these findings suggest that the level of BMP signaling, as
determined by local concentrations of ligands and antagonists, coordinately regulates cortical cell fate and morphogenesis. Defining the signaling pathways and gene regulatory events that correspond to
these effects should help elucidate the intracellular mechanisms of
developmental cell death, neurogenesis, and gliogenesis.
| |
FOOTNOTES |
Received Oct. 30, 1998; revised June 1, 1999; accepted June 4, 1999.
This work was supported by grants from the National Institutes of
Health (P.C.M., M.F.M., and J.A.K.). We thank Anthony Celeste, John
Wozney, and Genetics Institute for providing us with BMPs 2 and 4 and
the monoclonal antibody to BMP2. We thank Aris Economides, George
Yancopoulos, and Regeneron Pharmaceuticals for providing us with noggin
and anti-noggin antibodies.
Correspondence should be addressed to Dr. Peter C. Mabie, Department of
Neurology, Albert Einstein College of Medicine, Kennedy Center, Room
401, 1300 Morris Park Avenue, Bronx, NY 10461.
| |
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TOP
ABSTRACT
INTRODUCTION
