1 Laboratoire de Neurobiologie du Développement et de la
Régénération CNRS, 5 rue Blaise Pascal, 67000
Strasbourg, France
2 Department of Anatomy and Neurobiology, College of Medicine, University of
Vermont, Burlington, VT 05405, USA
3 GSF, Institute for Mammalian Genetics, Ingolstaedter Landstrasse 1, D-85764
Neuherberg, Germany
4 Massachusetts General Hospital Cancer Center, Department of Cell Biology,
Harvard Medical School, Charlestown, Massachusetts 02129, USA
5 Laboratoire d'embryologie cellulaire et moléculaire
Collège de France, 49B, Avenue de la Belle Gabrielle, 94736 Nogent sur
Marne, France
* Author for correspondence (e-mail: mohier{at}neurochem.u-strasbg.fr)
Accepted 10 December 2002
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SUMMARY |
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Key words: Neural stem cells, Notch-Delta signaling, Cell fate specification, Neurospheres, Mouse
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INTRODUCTION |
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Owing to the absence of specific markers, neural stem cells cannot be identified prospectively and their developmental behavior is therefore difficult to address in vivo.
As neurospheres are clonally derived from neural stem cells
(Weiss et al., 1996), they
provide a good experimental system for studying the mechanisms involved in the
proliferation and differentiation of these multipotent cells in
development.
The production of neurospheres relies on the selection of neural stem cells
from embryonic (or adult) brain through the action of EGF. In the presence of
EGF, neural stem cells proliferate and form clonally derived clusters of cells
floating in the medium that are referred to as neurospheres (or spheres). Each
neurosphere represents the clonal progeny of a neural stem cell; as such, it
consists of a heterogeneous population of cells, including the neural stem
cells themselves (representing less than 5% of the cells) and their progeny.
This progeny consists of uncommitted (early progenitors) as well as committed
(late) progenitor cells. These cells remain in an undifferentiated state until
they are induced to differentiate by providing a solid support, on which they
attach and give rise to neurons, oligodendrocytes and astrocytes in
reproducible proportions (Reynolds and
Weiss, 1992; Weiss et al.,
1996
).
The Notch signaling pathway has been shown to define a fundamental cell
interaction mechanism that influences cell fate decision by interaction
between cellular neighbors. The involvement of Notch signaling in neuronal
development has been extensively documented in invertebrates and its action
has been shown to be highly pleiotropic and indeed context dependent. During
neurogenesis, Notch has been shown to inhibit neuronal differentiation in many
organisms in vivo and in vitro (Fortini et
al., 1993, Struhl et al.,
1993
; Nye et al.,
1994
; Artavanis-Tsakonas et
al., 1995
; Artavanis-Tsakonas
et al., 1999
; Henrique et al.,
1997
). Notch activation has also been shown to suppress
oligodendrocyte development from oligodendrocyte precursor cells (OPCs)
(Wang et al., 1998
). Recently,
Notch signaling has been found to trigger the differentiation of several types
of glial cells, including radial glia
(Gaiano et al., 2000
), Schwann
cells (Morrison et al., 2000
),
Müller cells in retina (Furukawa et
al., 2000
) and astrocytes
(Tanigaki et al., 2001
;
Lütolf et al., 2002
).
In the present study, we used neurospheres to examine the involvement of
Notch in the generation, the maintenance and the differentiation of neural
stem cells by comparing neurospheres produced from mice embryos deficient for
Delta-like gene 1 (Dll1lacZ/lacZ)
(Hrabé de Angelis et al.,
1997), with wild type neurospheres produced from the littermate
controls. We find that the Dll1lacZ/lacZ mutation affects
neither the generation nor the maintenance of neural stem cells in vitro. By
contrast, the Dll1lacZ/lacZ mutation strongly affects the
developmental potential of neurospheres. Neurospheres prepared from
Dll1lacZ/lacZ mutant embryos display an increase in the
production of neurons at the expense of both oligodendrocytes and astrocytes.
This mutant phenotype could be rescued when Dll1lacZ/lacZ
spheres differentiated in the presence of wild-type spheres conditioned
medium. Temporal modulation of Notch activation by soluble forms of ligands
indicates that Notch acts in two steps. Initially, it controls a switch
between neuronal fate to glial fate, repressing neuronal fate and promoting
glial fate (including both astrocytes and oligodendrocytes). In a second step,
Notch affects the differentiation decisions of precursors already committed to
a neuronal or a glial lineage; it promotes the differentiation of astrocytes
while inhibiting the differentiation of both neurons and oligodendrocytes.
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MATERIALS AND METHODS |
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Neurospheres have been prepared as described by Tropepe et al.
(Tropepe et al., 1999).
Telencephalons of embryos (E10.5), were dissected and mechanically dissociated
in the serum-free neurosphere culture medium as described by Vescovi et al.
(Vescovi et al., 1993
)
[DMEM:F12 (1:1) supplemented with 25 µg/ml insulin, 100 µg/ml
transferrin, 20 nM progesterone, 60 µM putrescine, 30 nM selenium and 20
ng/ml EGF]. The dissociated cells were plated in 24-well plates (Nunc). After
7 days in vitro (DIV), most of the cells died, although a small percentage of
cells proliferated by forming clusters of undifferentiated cells floating in
the medium, referred to as the neurospheres (spheres). These primary spheres
were spun down (65 g for 5 minutes) and were dissociated
mechanically and chemically, making use of a `dissociation solution' (Sigma)
and were further expanded by transfer into fresh neurosphere culture medium in
which they were cultured for generating secondary spheres. Secondary cultures
were transferred into 250 ml flasks (Falcon). Culture flasks were coated with
poly(2-hydroxy-ethyl-methacrylate) (polyHEMA; Sigma, 1.6 mg/cm2) to
prevent cell attachment. Neurospheres could be maintained for long periods of
times by successive passages involving dissociation and proliferation, or were
frozen in neurosphere culture medium containing 10% DMSO, when necessary. Out
of seven Dll1lacZ/lacZ mutant embryos, three lines of
spheres could be obtained, two of which have been maintained. We checked that
these two lines behaved in the same way and showed similar differentiating
potentials (see below). Many of the reported experiments have been carried out
in the second line and showed similar results (data not shown).
J1EC and 3T3-conditioned medium
Conditioned medium enriched with a soluble form of human Jagged, was
produced from stably transfected NIH-3T3 cells
(Sestan et al., 1999).
HJaggedEC-3T3 cells, and untransfected NIH-3T3 cells (used as a control) were
cultured until confluence, in DMEM containing 10% FBS. When the culture became
confluent, the medium was removed and replaced by the neurosphere culture
medium for another 3 days. The cells were then harvested by centrifugation and
the resulting conditioned supernatant was filtered through a 0.22 µm filter
unit millex-GS (Millipore). This conditioned medium (referred to as
J1EC) was added undiluted to the Dll1lacZ/lacZ
spheres for various time windows during either the proliferation and/or the
differentiation phase(s), as described in the experimental diagrams.
Conditioned medium from untransfected NIH-3T3 cells (3T3) was used as a
control.
Differentiation and analysis of neurospheres
After various times of proliferation, 50-100 neurospheres were plated onto
polyornithine (poly-L-ornithine Sigma)-coated (14 mm) coverslips, in a 24
wells plate (Nunc) to differentiate. Differentiation of neurospheres is often
described as requiring the withdrawal of EGF, which is replaced by fetal
bovine serum (FBS) (Vescovi et al.,
1993, Tropepe et al.,
1999
). In order to avoid the hazardous effect of FBS, while
preserving cell survival, we reduced EGF concentration to 2 ng/ml during the
differentiation phase, which is sufficient to allow cell survival but
minimizes proliferation. Under these conditions, the differentiating sphere
remains a dynamic structure where generation of new cells (stem cells and
progenitors) is diminished but not totally abolished.
Notch activation experiments
Fully dissociated Dll1lacZ/lacZ spheres were grown in
J1EC-conditioned medium, (or in NIH-3T3 control medium) in 50 ml
flasks coated with polyHEMA. At various times, as indicated on the diagrams,
the growing spheres were collected by centrifugation, washed out in PBS, and
the incubation was continued either in 3T3 control medium or in fresh
J1EC. After the proliferation phase, neurospheres were harvested
and plated on polyornithine-coated coverslips either in J1EC or in
the control medium. After various times of differentiation, the samples were
processed for immunostaining for identification of the cell types.
Immunostaining
Neurospheres were fixed for 20 minutes in 4% paraformaldehyde in PBS (pH
7.4), washed in PBS and permeabilized 5 minutes with PBS/0.5% Triton X-100
(Sigma). The neurospheres were incubated overnight at 4°C in PBS
containing 3% BSA and the appropriate mixture of antibodies. Primary
antibodies used were mouse monoclonal anti-MAP2 (2a+2b) (1/400, Sigma)
specific for neurons, rabbit polyclonal anti-GFAP (1/600, Dako) for
astrocytes, and rabbit polyclonal PDGFR (1/500, Santa Cruz) and mouse
monoclonal anti-O4 (1/20, Boehringer) for oligodendrocytes precursors cells
(OPCs) and immature oligodendrocytes, respectively. After washing in PBS,
differentiating spheres were incubated for 1 hour with Cy2-, Cy3- and
Cy5-conjugated secondary antibodies (1/300 Amersham) or Alexa 488-conjugated
antibodies (1/1000, Molecular Probes). Preparations were counterstained with
TO-PRO (1/15000, Molecular Probes), mounted in Aquamount (Polyscience) and
viewed for triple immunofluorescence using a Zeiss LSM 410 confocal
microscope.
Quantitative results
Data are based on three or four independent experiments in which an average
of more than 10 spheres were analyzed per treatment, per experiment. Under the
conditions used for cell concentration after full dissociation
(1.105 cells/ml) and plating (50-100 spheres/coverslip), each
neurosphere may be considered as the clonal progeny of a single neural stem
cell. For cell type quantitative estimation, neurospheres were chosen of
approximately the same size. The confocal plane was at the basis of the
neurosphere, i.e. where the cells differentiate. The optical slice was 2
µm. The results are expressed in percentage of total cell number assessed
from TO-PRO staining. The graphed results are shown as means±s.e.m.
Group changes were assessed using oneway ANOVA. When statistical differences
were obtained between groups at P
0.05, multiple pair-wise
comparisons were made using the Turkey-Krammer method. In the text, P
indicates statistical significant differences.
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RESULTS |
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The early death of Dll1lacZ/lacZ mutant embryos (around day 12) prevented further analysis of the role of Dll1 in the maintenance of neural stem cells in vivo. In vitro, we observed no difference in the maintenance of mutant and wild-type neurospheres. Based on the neurosphere assay, the estimation of neural stem cell percentage from the dissociated neurospheres cellular population was similar in mutant and wild-type neurospheres, and was below 5% of total cell number, showing little variation through successive passages (data not shown). These observations suggest that wild type and Dll1lacZ/lacZ neural stem cells behave approximately the same, undergoing the same number of symmetrical and asymmetrical divisions.
Dll1lacZ/lacZ mutant spheres display an increase
in neurons at the expense of glial cells
After verifying that wild-type neurospheres differentiated according to a
pattern qualitatively and quantitatively reproducible under the culture
conditions employed, we compared the differentiation potential of
Dll1lacZ/lacZ and wild-type neurospheres. The general
experimental protocol is schematized in
Fig. 1. Three-day-old spheres
were plated on polyornithine in the presence of 2 ng/ml of EGF, and allowed to
differentiate for various times depending on the temporal pattern of
expression of the markers employed for immunocytochemistry. The percentage of
each of the cell types was determined by triple immunostaining, including a
combination of two specific markers (in addition to TO-PRO, a marker for
nuclei), and analyzed by confocal microscopy. Cell types were identified using
antibodies against MAP2 for neurons, against GFAP for astrocytes, against
PDGFR for oligodendrocyte precursor cells (OPCs)
(Hutchins, 1995) and against
O4 for immature oligodendrocytes.
|
After 5 hours on polyornithine (Fig. 2A), a few cells in wild-type spheres started expressing MAP2. Low production of neurons (which do not exceed 10% of total cell number), is a characteristic of wild-type neurospheres (Fig. 2B). By contrast, GFAP-positive cells which appear after 48 hours on polyornithine, represent more than 50% of total cell number and indicate that astrocytes are the major cell type generated from wild-type neurospheres (Fig. 2A, part V). Oligodendrocytes are difficult to quantify accurately because of the aspect of the O4 marker (Fig. 2VII); however, PDGFR-positive cells indicate that OPCs represent about 15-20% of total cell number (Fig. 2B). In keeping with the expression of PDGFR, after 6 days on polyornithine, many cells in wild-type neurospheres continue to express O4, indicative of an oligodendroglial lineage commitment. Note, however, that these O4-positive cells exhibit no processes and appear therefore as morphologically poorly differentiated (Fig. 2A, part VII).
|
Contrasting with wild-type spheres, Dll1lacZ/lacZ mutant spheres show an increase in MAP2-expressing cells, which represent more than 50% of total cell number, indicating that neurons are the major cell type generated by Dll1lacZ/lacZ mutant spheres. This increase in neurons takes place at the expense of both glial lineages as evidenced by the decrease in GFAP-positive cells (Fig. 2A, parts V,VI) as well as in PDGFR-positive cells (Fig. 2A, parts III,IV), indicating a decrease in astrocytes and OPCs, respectively. Interestingly, the few remaining O4-expressing cells exhibited many processes, suggesting that they were morphologically more differentiated than in the wild-type neurospheres (Fig. 2A, parts VII, VIII). Note that, together, neurons, oligodendrocytes and astrocytes did not amount to 100%, consistent with the observation that many cells in the core of the sphere failed to express any marker, and were likely to correspond to early uncommitted precursors (Fig. 2B). We sought to determine whether the alteration in the differentiation potential of wild-type and Dll1lacZ/lacZ mutant spheres could be accounted for by Notch selectively acting on the different lineages by affecting their survival or proliferation.
In order to examine whether the quantitative variations between wild-type and Dll1lacZ/lacZ mutant phenotypes might be due to selective apoptosis, we searched for dying cells in individual spheres, making use of TUNEL analysis, counterstained with TO-PRO, at various times after the induction of differentiation. Up to 2 days on polyornithine, the number of apoptotic cells remained low, representing less than 5% of total cells. For longer periods of differentiation, it increased (up to 15% after 5 days on polyornithine). No significant difference was found, however, between wild-type and Dll1lacZ/lacZ neurospheres (data not shown). Pulses of BrdU incorporation, combined to either MAP2 or GFAP, in wild-type versus Dll1lacZ/lacZ spheres showed no difference between the two lineages, eliminating the possibility that the higher number of neurons seen in Dll1lacZ/lacZ mutant spheres was the result of an increased lineage specific proliferation event (data not shown).
The examination of the Dll1lacZ/lacZ phenotype showed that the lack of Dll1 activity, which presumably results in the inability to activate the Notch receptor, is consistent with the previous finding that Notch signaling represses neurogenesis, and the more recent finding that Notch promotes the generation of astrocytes. Despite the difficulty to quantify the oligodendrocytes, we think that our results are consistent with the notion that Notch promotes the OPCs production (from a quantitative estimation of PDGFR-positive cells), while it inhibits their further differentiation into more mature structures (from a qualitative estimation of morphological changes of O4-positive cells).
Dll1 has a gene-dosage effect on the differentiation potential of
neural stem cells
Heterozygous Dll1lacZ/+ mouse embryos are
phenotypically normal (Hrabé de
Angelis et al., 1997; Beckers
et al., 1999
). After 24 hours on polyornithine, neurospheres
heterozygous for Dll1 showed a twofold increase in the number of
MAP2-positive cells, but no variation in the number of GFAP-positive cells
(data not shown). After 3 days of differentiation
(Fig. 3A,B), the increase in
neurons was confirmed (from 20.3±4.3% to 27.7±3.1%;
P
0.01) and as in homozygous Dll1lacZ/lacZ
neurospheres, this increase seemed to take place at the expense of astrocytes
(from 50.3±6.9% to 23.2±4.6%; P
0.001), even though
the increase in neurons does not quantitatively compensate for the decrease in
astrocytes. This quantitative variation was accompanied by a clear
morphological modification, with neurons exhibiting longer processes, whereas
astrocytes appeared with thinner processes and lacking the star-like
morphology characteristic of mature astrocytes.
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J1EC rescues the DII1lacZ/lacZ mutant
phenotype with thresholds different for neurons and astrocytes
It is well established that Drosophila Notch is acting through
interaction with membrane-bound ligands, including Delta
(Vassin et al., 1985) and
Serrate (Fleming et al., 1990
).
However, recent data have shown that under particular circumstances,
invertebrate and vertebrate Notch activation can be mediated by apparently
soluble forms of ligands that are found in the conditioned medium of ligand
expressing cells (Klueg et al.,
1998
; Qi et al.,
1999
; Sestan et al.,
1999
). We found that it was indeed possible to `rescue' the mutant
Dll1lacZ/lacZ spheres to differentiate normally in the
presence of wild-type conditioned medium (WTCM) (data not shown).
The Dll1-dependent activity present in the WTCM, provided us with a tool for temporally modulating Notch activation, making it possible to analyze the effect of Notch signaling at various steps of the complex differentiation process leading from the neural stem cell to the differentiated cell types. However, repeated experiments showed an inconsistency in the potency of the WTCM to trigger the rescue of the Dll1lacZ/lacZ mutant phenotype. We were thus obliged to resort to a more reliable source of Notch ligand.
We used NIH-3T3 cells transfected with the human jagged extracellular
domain (hJagEC-3T3 cells), which was shown to be released in the medium.
Conditioned medium from these cells (hereafter referred to as
J1EC), has been shown capable of regulating neurite outgrowth and
Notch-dependent gene expression (Sestan et
al., 1999). Jagged 1 is the vertebrate counterpart of Serrate, the
second Notch ligand in Drosophila. Jagged and soluble forms thereof,
like Delta, have been shown to activate the Notch receptor
(Lindsell et al., 1995
;
Shimizu et al., 2000a
;
Shimizu et al., 2000b
;
Solecki et al., 2001
;
Varnum-Finney et al.,
1998
).
In order to check whether a soluble form of jagged 1 product could rescue Dll1lacZ/lacZ in the same way as WTCM, 3-day-old Dll1lacZ/lacZ mutant neurospheres were exposed to dilutions (up to 20-fold) of J1EC during the differentiation period and were further analyzed by immunostaining for their differentiation capacities in comparison with mutant spheres treated with untransfected NIH-3T3 cell-conditioned medium (referred to as 3T3) used as a control, as described in Fig. 3C. Consistent with the effects observed with WTCM, the results (Fig. 3D,E) showed that addition of J1EC resulted in a decrease in the number of differentiated neurons (a 20-fold dilution was necessary to abolish this effect, data not shown). The decrease in neurons was accompanied by an increase in astrocytes for only higher concentrations of J1EC (Fig. 3D, compare parts II, III and IV). Quantitative data show little variation in the number of neurons and astrocytes in response to decreasing doses of J1EC (for neurons, from 36.1 to 10.8, 10.9 and 10.5 for the three dilutions of J1EC, respectively; for astrocytes, from 12.5 and 12.6 under non rescuing conditions, to 43.2 and 38.5 for higher concentrations of J1EC). These data suggest that for the tested dilutions, the response is all or nothing, with different thresholds for neurons and astrocytes. In addition, few GFAP-positive cells that were generated in response to low concentrations of J1EC (Fig. 3D, part IV), exhibited a poorly differentiated morphology, reminiscent of that observed in Dll1lacZ/+ neurospheres (Fig. 3A).
Notch activation represses both neuronal specification and
differentiation
We investigated further the effect of Notch activation on
Dll1lacZ/lacZ spheres by treating them with
J1EC or control (3T3) for different lengths of time. The results
are shown in Figs
4,5,6.
Exposure of Dll1lacZ/lacZ mutant spheres to
J1EC resulted in all cases in a decrease in the production of
neurons, in comparison with Dll1lacZ/lacZ spheres treated
with 3T3-conditioned medium used as a control. The strongest effect was
observed when J1EC was provided during both the proliferation and
differentiation phases (Fig. 4A-C, parts
I,II; from 47.9±2.0% to 10.3±0.8%;
P0.001). These data are consistent with the previous observations
indicating that Notch inhibits neurogenesis
(Fortini et al., 1993
;
Struhl et al., 1993
;
Nye et al., 1994
;
Artavanis-Tsakonas et al.,
1995
; Artavanis-Tsakonas et
al., 1999
; Henrique et al.,
1997
).
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A decrease in the proportion of neurons was not only seen when
J1EC was provided during the proliferation phase
(Fig. 4A-C, parts I, III; from
47.9±2.0 to 18.8±1.8%; P0.001), but also when it
was provided transiently during the first 24 hours at the initiation of
proliferation and then washed out (Fig.
4A-C, part IV; 12.7±1.7%; P
0.001). In fact, an
exposure to J1EC for only 5 hours was sufficient to repress
neurogenesis significantly (data not shown), whereas a treatment for 3 hours
was not enough (see Fig. 6A-C, parts I,
III; from 47.8±2.3% to 40.4±2.1%; n.s.,
P>0.05). These results are consistent with earlier findings in the
PNS neural crest stem cells (Morrison et
al., 2000
) and suggest that a transient activation of Notch is
capable of repressing the neurogenic potential of neural stem cells. Moreover,
it appears that this inhibition was irreversible, as neurogenesis did not
resume after J1EC removal.
We found that treatment of Dll1lacZ/lacZ spheres with
J1EC during the differentiation phase also resulted in a decrease
in neurons (Fig. 4A-C, part
I,V; from 47.9±2.0% to 8.0±0.7%;
P0.001). This observation suggests that precursors that were
allowed to adopt a neuronal fate in response to the Notch inactivity during
the proliferation phase, were prevented from further differentiating into
MAP2-positive cells upon Notch activation during the differentiation phase.
Together, these data suggest that Notch signaling inhibits neuronal
differentiation not only by preventing uncommitted precursors to acquire a
neuronal fate but also at a later stage, when precursors which were already
committed to a neuronal fate, will take on the decision to differentiate into
MAP2-positive neurons.
Notch activation promotes the specification and accelerates the
differentiation of astrocytes
The effects of Notch activation on the production of astrocytes can be
inferred from the same experiment (Fig.
4), which shows that the decrease in neurons is generally
accompanied by an increase in the number of GFAP-expressing cells. This
gain-of-function effect is opposite to the Dll1lacZ/lacZ
phenotype, where the inability to activate the receptor (loss of function)
resulted in an increase in neurons (compared with wild-type spheres) at the
expense of astrocytes. Together, these observations suggest that neurons
versus astrocytes define a developmental decision controlled by Notch.
However, we found that a transient exposure to J1EC for only 24
hours, even though it was reproducibly sufficient to induce a decrease in
neurons, was not always accompanied by a substantial increase in astrocytes
(Fig. 4A-C, part IV). Exposure
to J1EC for longer times, reproducibly resulted in an increase of
the production of mature astrocytes (Fig.
4A-C, parts I, II; from 11.2±1.1% to 22.2±1.9%;
P0.001), and to a lesser extent in
Fig. 4A-C, part V (from
11.2±1.1% to 17.2±1.0%; P
0.05).
These experiments were repeated under narrower time intervals on both
Dll1lacZ/lacZ mutant and wild-type spheres
(Fig. 5). The results showed
that an activation time of 24 h after dissociation was sufficient to repress
differentiation of neurons (from 35.2±2.2% to 9.96±1.3%;
P0.001, Fig. 5A-C, parts
I,II) but not to trigger GFAP expression (12.3±0.8% and
10.0±0.9%; not significant P>0.05). By contrast, when the
activation took place later and for longer time periods (from d-1 to d+2,
Fig. 5A-C, part III), GFAP-positive astrocytes increased up to more than 55% of total cell number.
These results could be correlated to J1EC dilution experiments
(Fig. 3C,D) and showed that the
decrease in neurons was not necessarily accompanied by a correlative increase
in astrocytes; these two phenomena could be disconnected by the temporal
modulation of Notch function (transient activation during the proliferation
phase), as well as by lowering the concentration of the inducing signal during
the differentiation phase. We assume that these `disconnecting' conditions
generate non-differentiated cells which appear as immunonegative in the
neurospheres and which may account for the strong decrease of the added
percentage of neurons and astrocytes under the mutant and rescuing conditions
(for example in Fig. 4A-C, from
59.1% in part I versus 20-30% in parts II-V). These cells are likely to
undergo cell death by apoptosis as usually described for cells which were
misdirected and do not differentiate properly
(Lütolf et al., 2002
).
TUNEL analysis carried out under these experimental conditions showed an
increase in TUNEL-positive cells with time (from 5.3% after 24 hours on
polyornithine to 13.3% after 5 days of differentiation); however, no
difference could be observed between the different experimental conditions,
suggesting that apoptosis resulting from the `misdirection' of cell lineage,
could not be distinguished from apoptosis normally occurring in neurospheres
differentiating in serum-free medium.
The early as well as late activation of Notch in wild-type spheres led to an increase in the number of GFAP-positive cells (Fig. 5A-C, parts IV, V, VI; from 47.5±4.4% to more than 80%) in both cases. In addition, GFAP-positive cells displayed a different morphology, indicative of a more mature state of development (a similar phenotype of wild-type spheres normally requires at least 5 days on polyornithine). These data suggest that the activation of Notch in wild-type spheres not only increases the number of astrocytes but also their rate of production and differentiation. We also noted that this increase in astrocytes was never accompanied by a total suppression of neurons (Fig. 5A-C, parts V,VI).
Notch activation promotes the production of OPCs and inhibits their
subsequent differentiation into mature oligodendrocytes
Examination of the Dll1lacZ/lacZ neurospheres showed
that lack of Dll1 activity resulted in a decrease in the number of
PDGFR-expressing cells. Paradoxically, however, the few resultant O4-positive
cells exhibited a well differentiated morphology when compared with poorly
differentiated wild-type oligodendrocytes
(Fig. 2A-C, parts VII, VIII). Together, these observations suggested that Notch signaling regulates the
production of oligodendrocytes in two steps: first, by promoting the
production of OPCs; and second, by inhibiting the differentiation of
O4-expressing cells into more mature oligodendrocytes. This statement predicts
that a transient treatment of Dll1lacZ/lacZ neurospheres
with J1EC would result in an increase in PDGFR-positive cells and
in their increased capacity to differentiate.
The positive effect of Notch signaling on the production of OPCs was
observed when Dll1lacZ/lacZ spheres were transiently
exposed to J1EC during the proliferation protocol and resulted in a
significant increase in PDGFR-positive cells
(Fig. 6A-C, parts I, II; from
6.64±1.8% to 25.3±2.5%; P0.001). When
Dll1lacZ/lacZ spheres were constantly treated with
J1EC during both proliferation and differentiation
(Fig. 6A-C, part V), a large
number of O4-expressing cells were observed (even though no precise
quantitative estimation could be made), indicating that most of the
PDGFR-positive cells turned into O4-positive cells, thereby suggesting that
this process is not inhibited by Notch activity. However, these cells appeared
as poorly differentiated, with morphology reminiscent of O4-positive cells in
wild-type spheres (Fig. 2A-C, part
VII). By contrast, when Dll1lacZ/lacZ
neurospheres were transiently exposed to J1EC (during the
proliferation phase), a large number of O4-positive cells were observed, some
of which displayed a rather differentiated morphology (compare parts V and VI
in Fig. 6A-C). Unfortunately,
this assessment cannot be further substantiated by the use of markers for
mature oligodendrocytes, such as MAG or MBP, as our cultures die before the
oligodendrocytes can reach such a degree of maturation.
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DISCUSSION |
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Neurospheres recapitulate many of the pleiotropic effects of Notch
signaling on neurogenesis
Dll1lacZ/lacZ mutant-derived neurospheres show that
Notch signal modulation affects qualitatively and quantitatively the outcome
of neural stem cell differentiation. The differentiation phenotype of
Dll1lacZ/lacZ mutant spheres compared with that of
wild-type spheres, exemplifies many earlier findings of Notch effects on
neurogenesis: (1) Notch signaling inhibits neurogenesis
(Fortini et al., 1993;
Struhl et al., 1993
;
Nye et al., 1994
;
Artavanis-Tsakonas et al.,
1995
; Artavanis-Tsakonas et
al., 1999
; Henrique et al.,
1997
); (2) Notch signaling represses oligodendrocyte
differentiation from OPCs (Wang et al.,
1998
), whereas recently it has been found to promote the
differentiation of astrocytes (Tanigaki et
al., 2001
; Lütolf et al.,
2002
). Although many of these data were obtained from systems
comprising essentially one single cell type, neurospheres provide a global
insight of Notch function on all three major cell types comprising the CNS, in
an integrated system, making possible the analysis of their interactions.
Neural stem cells differentiate in response to Notch signaling with
different sensitivity for neurons and astrocytes
Gene-dosage effect can be inferred from the phenotype of
Dll1lacZ/+ heterozygous neurospheres showing a similar
(but weaker) phenotype of differentiation to that of homozygous neurospheres
(Fig. 3A,B). We note, however,
that the percentage of astrocytes in heterozygous spheres is strongly
decreased (from 50.3% in wild type to 23.2%), while the increase in neurons is
restrained (from 20.3% in wild type to 27.7% in
Dll1lacZ/+). This observation indicates that half a dose
of Dll1 in heterozygous spheres is still sufficient to partly repress
neurogenesis, although insufficient to promote astrocytic differentiation.
This statement is further corroborated by the dilution experiment
(Fig. 3D,E) where all dilutions
of J1EC result in the decrease in neurons, whereas the exogenous
ligand triggers the appearance of GFAP-positive cells only for the highest
concentrations. Together these results suggest that the inhibition of neurons
is more sensitive to ligand induction than the promotion of astrocytes. The
quantitative analysis of the results indicates that the response is
`all-or-nothing' with different thresholds for neurons and astrocytes. A
molecular support of these observations has been provided with the recent
finding that Ngn1, a neural bHLH gene activated downstream of
MASH1, was shown to inhibit directly the transcription of the
astrocyte marker gene, Gfap, in a mechanism independent of its effect
to promote neuronal differentiation (Sun
et al., 2001). It is thus conceivable that a decrease in ligand
concentration would induce a modulation in Ngn1 production and would
subsequently result in distinct and separate effects of Ngn1 in activating
neuronal differentiating genes and suppressing glial-specific genes. The
finding that astrocytes are requiring more ligand to achieve differentiation
may account for the variation of the percentage of astrocytes from one
experiment to the other (compare astrocytes in Figs
3,4,5)
and may be attributed to the variation in the efficiency of the rescuing
agent.
Neuronal versus glial lineage defines a developmental decision
controlled by the Notch pathway
Together the analysis of the Dll1lacZ/lacZ mutant
differentiation phenotype and the time-dependent modulation of the Notch
pathway are consistent with the tentative model of lineage tree of neural stem
cells described in Fig. 7.
|
We found that cells expressing neuronal markers are present in the small
spheres that underwent few cells divisions. Moreover, a few neurons were
always found in differentiating neurospheres, even under conditions where
Notch was overactivated by addition of exogenous J1EC to wild-type
neurospheres. Together, these observations suggest that: (1) in neurospheres,
neurons are generated before glia and represent therefore the primary fate of
neural stem cells; (2) cells at the origin of neurospheres maintain their
multipotency even after extensive ex vivo expansion (some of our neurosphere
lines are more than two years old), contrary to the progressive loss of
neurogenic capacity described for neural crest stem cells
(Morrison et al., 2000;
Kubu et al., 2002
); (3) P1
precursor, issued from the first asymmetrical division of the neural stem
cell, adopts a neuronal fate and may be recalcitrant to exogenous Notch
signals.
By contrast, our model predicts that Notch can be activated in P2, which, as a result, would be prevented from adopting a neuronal fate. We anticipate that P1, which is a neuronal precursor, is likely to express Dll1, thus providing the signal capable of activating Notch in P2, thereby suppressing its neuronal fate. In the absence of reliable markers for Notch ligands, it is difficult to further argue this hypothesis.
The observation that the transient activation of Notch in
Dll1lacZ/lacZ spheres is sufficient to inhibit the
production of neurons, and that this production does not resume upon removal
of the ligand, suggests that Notch activation in P2 causes an apparently
irreversible loss of neuronal potential. P2 is therefore committed to a glial
fate instead of being maintained in an undifferentiated and multipotential
state. This is consistent with the finding that in the PNS, transient
activation of Notch in the neural crest stem cells was sufficient to cause an
irreversible loss of neurogenic capacity accompanied by an accelerated glial
differentiation (Morrison et al.,
2000).
By contrast, our data are inconsistent with the recent finding that Notch
signaling does not appear to have a role in the neuronal/glial fate switch
(Hitoshi et al., 2002). Beside
the trivial explanation that this discrepancy was due to differences in the
experimental procedures, we believe it is more likely to be due to the fact
that these authors were specifically addressing Notch1 behavior. This
interpretation is further supported by recent experiments involving
conditional ablation of Notch1 in neurospheres (V. Taylor, personal
communication) and by our own results showing that neurospheres originating
from embryos heterozygous for Notch1 (contrary to
Dll1lacZ/+ spheres) show no quantitative modification in
the proportion of neurons/astrocytes. (J.B. and E.M., data not shown). This
assumption also suggests that the manipulation of each of the ligands (Dll1 or
Jagged1) we are describing in the present study is likely to affect more than
the Notch1 receptor.
Notch signaling controls the differentiation decisions of precursors
already committed to a neuronal or glial lineage
As a result of Notch function, precursors are generated that are fated
either to a neuronal (P1) or a glial fate (P2). However, these precursors do
not necessarily give rise to the more mature cell type that expresses the
appropriate differentiation marker. The experimental temporal modulation of
Notch activity is consistent with the notion that neuron precursors, as well
as glial precursors, could be blocked in a non-differentiating state, and that
their further differentiation depends on secondary Notch signaling.
Neuronal precursors that were normally generated in Dll1lacZ/lacZ mutant spheres, owing to the absence of Notch activity during the proliferation phase, do not develop into MAP2-expressing cells when Notch is activated during the differentiation phase (Fig. 4V).
On the contrary, precursors that were fated to a glial cell type upon
transient activation of Notch will not differentiate into GFAP-expressing
astrocytes (Fig. 4B, parts III and
IV; Fig. 5B, part
II) unless Notch is re-activated through the presence of soluble
ligand during the differentiation phase. We assume that these cells which were
blocked in a non-differentiated state, are likely to undergo cell death by
apoptosis, as usually described for cells that were misdirected and do not
differentiate properly (Lütolf et
al., 2002).
In keeping with its role in the specification of cell types, Notch is
positively acting for the differentiation of astrocytes and negatively acting
for the differentiation of neurons. By contrast, Notch signaling has two
contradictory effects on the production of oligodendrocytes. In a first step
it acts positively to promote OPC production, whereas it negatively regulates
their subsequent differentiation into oligodendrocytes; however, only the
latter effect has been previously reported in other systems that were already
committed to the oligodendroglial lineage
(Wang et al., 1998;
Kondo and Raff, 2000
).
Our model postulates that P2 is restricted to a glial fate with the potential to differentiate into either astrocytes or oligodendrocytes. Owing to the absence of specific markers, P2 cannot be identified in neurospheres. The existence of such a precursor with both astrocytic and oligodendroglial potential is controversial in vivo. The OPCs (formally called 0-2A) have long been investigated and have been shown to differentiate in vitro (in the presence of 10% FBS) into both oligodendrocytes and type II astrocytes that are positive for both GFAP and A2B5. We never observed cells with characteristics of type II astrocytes. P2 is therefore different from PDGFR cells, which, we assume, are already committed to an oligodendroglial lineage and are likely, under the conditions employed, to give rise only to 04-expressing oligodendrocytes.
Unfortunately, GFAP is likely to be a marker of astrocyte maturation rather
than of lineage commitment, thereby hindering the direct comparison of OPCs
with astrocyte precursors regarding Notch signaling. However, our observations
show that in no case were oligodendrocytes and astrocytes mutually exclusive
regarding Notch activation. We therefore conclude that the segregation between
oligodendrocyte and astrocyte lineages is independent of Notch signaling and
might derive from another mechanism, involving for example the transcription
factors OLIG1 and OLIG2 (Zhou and
Anderson, 2002).
It is clear that further experimentation will be necessary to test the validity of the differentiation model we propose. It is also clear, however, that Notch signals seem to play an important role in the differentiation of the neural stem cells lineages. Further analysis of the exact role that Notch signals play in neural stem cells will not only provide insights into the biology and underlying mechanisms of these cells but also provide a potential tool for manipulating their fate for therapeutic purposes.
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ACKNOWLEDGMENTS |
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