Unité 368 de l'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
* Author for correspondence (e-mail: charnay{at}wotan.ens.fr)
Accepted 27 August 2003
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SUMMARY |
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During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. We show that antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis.
Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilisation of the committed state. Finally, gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS.
Key words: Ebf1/Ebf genes, Neurogenesis, Cell cycle, Neuronal differentiation, HLH domain
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Introduction |
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Another class of genes, those of the Ebf/Olf1 family (reviewed by
Dubois and Vincent, 2001;
Liberg et al., 2002
), has been
involved in the control of neuronal differentiation.
Ebf/Olf1 was independently identified as a transcription
factor implicated in mouse B-lymphocyte differentiation (Ebf)
(Hagman et al., 1993
;
Travis et al., 1993
) and in
transcriptional control of the rat olfactory marker protein gene
(Olf1) (Kudrycki et al.,
1993
; Wang and Reed,
1993
). Three other rodent genes showing high similarity to
Ebf (renamed Ebf1), Ebf2 (also known as
Mmot1 or O/E-3), Ebf3 (also known as
O/E-2) and O/E-4, were subsequently identified
(Garel et al., 1997
;
Malgaretti et al., 1997
;
Wang et al., 1997
;
Wang et al., 2002
). In other
species, Ebf orthologs have been found in C. elegans, Drosophila,
Xenopus and zebrafish (Crozatier et
al., 1996
; Bally-Cuif et al.,
1998
; Dubois et al.,
1998
; Prasad et al.,
1998
; Pozzoli et al.,
2001
). All Ebf proteins share an atypical non-basic
helix-loop-helix motif (HLH), with an additional type 2 helix and a novel zinc
coordination motif. This latter motif is involved in DNA binding, which also
requires homodimerisation or heterodimerisation with other family members
mediated by the HLH structure (Hagman et
al., 1995
). A detailed analysis performed in the mouse revealed
that Ebf1, Ebf2 and Ebf3 are expressed along the entire
rostro-caudal axis of the developing CNS, with overlapping patterns, except in
the forebrain where each of them is restricted to specific regions
(Garel et al., 1997
;
Wang et al., 1997
). Expression
is observed in differentiating neurones, Ebf2 being restricted to
early post-mitotic neurones whereas Ebf1 and Ebf3 expression
is maintained in more differentiated cells. These data suggest a general role
for Ebf genes in neuronal differentiation. In accordance with this,
overexpression of a dominant-negative mutant of Xenopus Ebf2,
Xcoe2/XEbf2, prevents primary neurone differentiation in Xenopus
embryos (Dubois et al., 1998
).
Both XEbf2 and XEbf3 were shown to have neurogenic activity
in this system, but were proposed to be involved in different steps of the
neurogenic cascade. Whereas XEbf2 acts at an early stage during the
commitment process, downstream to XNgn1 but upstream of XNeuroD,
XEbf3, which is expressed later, functions downstream to
XNeuroD, in neuronal differentiation
(Dubois et al., 1998
;
Pozzoli et al., 2001
). In the
mouse, analysis of Ebf1/ mutants revealed
neuronal differentiation defects in CNS regions where Ebf1 is the
sole family member to be expressed. Hence, in the striatum, post-mitotic
neurones that leave the subventricular zone (SVZ) en route to the mantle layer
appear unable to downregulate genes normally restricted to the SVZ, or to
activate some mantle-specific genes (Garel
et al., 1999
). In the hindbrain, facial branchiomotor neurones
show an abnormal migratory pathway, presumably as a result of incorrect
interpretation of environmental guiding cues
(Garel et al., 2000
). A
migration defect was also observed in gonadotropin-releasing
hormone-synthesizing neurones in Ebf2/ mice
(Corradi et al., 2003
). These
latter animals also show peripheral nerve defects, although it was not
established whether this is of central and/or peripheral origin. Altogether
these studies confirm that the Ebf genes play important and different roles in
neurogenesis, and suggest that significant redundancy exists between them.
Although work performed in Xenopus has provided information on the position of Ebf genes in the neurogenic cascade, it was restricted to primary neurogenesis in the ectoderm, which significantly differs from neurogenesis in higher vertebrates, in which neurones are continuously generated in the proliferative neuroepithelium and migrate to the pial surface when they differentiate. Furthermore, in gene targeting experiments in the mouse, compensation between the different Ebf genes is likely to have prevented the unravelling of important aspects of their function. This prompted us to undertake a study of Ebf gene function in neurogenesis by electroporation of the chick embryo neural tube, where we can use both gain- and loss-of-function approaches in a higher vertebrate. We provide evidence that Ebf genes play general and essential functions in the neurogenic process in higher vertebrates, acting downstream to proneural genes. They are necessary for initiation of both migration toward the mantle layer and neuronal differentiation, but are not required for cell cycle exit, and thus allow the uncoupling of these two aspects of neurogenesis.
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Materials and methods |
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PCR-amplified DNA fragments and cloning junctions were checked by
sequencing. cDNA clones corresponding to chick Ebf1 and Ebf3
DNA binding domains were obtained essentially as described
(Garel et al., 1997), using
5-day-old whole chick embryo total RNA. Degenerated oligonucleotide primers
corresponding to the conserved amino acid sequences AHFEKQP and DNMFVHNN that
flank the Drosophila collier and mouse Ebf1 DNA binding domains were
as described previously (Garel et al.,
1997
). GenBank Accession numbers for chick Ebf1 and
Ebf3 clones are AY270034 and AY270035, respectively.
In ovo electroporation
Electroporations were carried out as described
(Itasaki et al., 1999;
Giudicelli et al., 2001
).
Briefly, fertilised eggs were incubated at 37°C to the indicated embryonic
stages. DNA in 10 mM Tris (pH 8.0) was injected into the neural tube at the
following concentrations:
Ebf, 2 µg/µl; reporter
plasmids [pAdRSVßgal (Le Gal La Salle
et al., 1993
) and pEGFP-N1 (Clontech)], 0.5 µg/µl; other
constructs, 1.5 µg/µl. The total DNA concentration in each
electroporation experiment was kept constant by adding vector plasmid DNA.
Embryos were electroporated at hindbrain or thoracic spinal cord levels.
Electroporations were performed with a BTX820 electroporator (Quantum) with
the following parameters: hindbrain level, 4 pulses of 25 V and 40
milliseconds at a frequency of 1 Hz; spinal cord level, 6 pulses of 25 V and
50 milliseconds at a frequency of 1 Hz. To evaluate the efficiency of
electroporation, a GFP expression vector (pEGFP-N1) was systematically
co-transfected in embryos to be processed for in situ hybridisation or
peroxidase-revealed immunohistochemistry, and only efficiently electroporated
embryos were analysed. Following electroporation, the eggs were incubated for
10 to 40 hours, as indicated. The embryos were harvested in phosphate buffered
saline (PBS), fixed in paraformaldehyde (PFA, 4% in PBS) for 3 hours for
immunohistochemistry, or for more than 6 hours for in situ hybridisation, and
dehydrated in a methanol series. For ß-galactosidase detection, the
embryos were fixed for 20 minutes and stained with X-gal as previously
described (Schneider-Maunoury et al.,
1993
).
In situ hybridisation
In situ hybridisation with digoxigenin-labelled riboprobes on whole-mount
embryos or vibratome sections was performed as described
(Wilkinson and Nieto, 1993)
and double in situ hybridisation was as described previously
(Giudicelli et al., 2001
). In
this latter case one of the probes was labelled with fluorescein-UTP.
Digoxigenin and fluorescein were detected sequentially with alkaline
phosphatase-coupled antibodies (Roche). NBT/BCIP (purple) staining was carried
out first, typically on digoxigenin-labelled probes. After removal of the
antibody, INT/BCIP (orange, red) staining was performed on
fluorescein-labelled probes. After whole-mount in situ hybridisation, embryos
were flat-mounted or microtome sectioned. For vibratome section (50 µm
thick), embryos were embedded in 4% agarose or albumin/gelatine. The chick
probes were as follows: cEbf1, cEbf3, N-cadherin and CRABPI
(this work); R-cadherin (Inuzuka
et al., 1991a
); Cash1
(Jasoni et al., 1994
);
Ngn1 and Ngn2 (Perez et
al., 1999
); Islet1, Islet2 and Lim1
(Tsuchida et al., 1994
).
Cell proliferation and cell apoptosis analyses
Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation.
BrdU, 15% (w/v) in PBS, was injected into the lumen of the neural tube and the
embryos were harvested 2 hours later. BrdU immunodetection was performed on
sections treated with 2 N HCl, 0.5% Triton X-100 in PBS for 30 minutes at
37°C, after blocking and before incubation with primary antibodies.
Apoptosis was detected by fluorescein labelling of DNA strand breaks (TUNEL,
Roche) on 50 µm-thick vibratome sections.
Immunohistochemistry
Immunohistochemistry was normally revealed by fluorescence, except for the
detection of neurofilaments in flat-mounted hindbrains. In this latter case,
we used a biotinylated hamster antibody directed against mouse IgG (Vector,
1:400) and streptavidin-horse radish peroxidase (Amersham, 1:500), to detect
the anti-neurofilament antibody. Peroxidase activity was revealed with
diaminobenzidine (Sigma), in the presence of nickel ammonium to enhance the
staining. For immunofluorescence, 50 µm-thick vibratome sections were
prepared from embryos embedded in 4% agarose, and blocked in PBS containing
0.25% Triton X-100 and 5% donkey serum. Primary antibodies were incubated in
the same solution overnight at 4°C. Incubations with the secondary
antibodies were at room temperature for 2 hours. All washes were performed
with PBS containing 0.25% Triton X-100. Sections were mounted in Vecta Shield
(Vector). Immunofluorescence pictures were acquired on a Leica TCS 4D confocal
microscope and assembled with Adobe Photoshop. Antibodies and dilutions were
as follows: neurofilaments, mouse monoclonal 3A10 (1:20, Developmental Studies
Hybridoma Bank (DSHB)); ß-tubulin-type III (Tuj1), mouse monoclonal and
rabbit polyclonal (1:500, Babco); BrdU, mouse monoclonal (1:100, Becton
Dickinson); ß-galactosidase, rabbit polyclonal (1:700 Cappel); Islet 1/2,
mouse monoclonal 39.4D5 (1:100, DSHB); HA, rat monoclonal (1:400, Roche);
Flag, rabbit polyclonal (1:200, Sigma); FITC-, Cy3- and Cy5-conjugated
secondary antibodies (1:200-1:800, Jackson Immuno Research).
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Results |
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In situ hybridisation analysis of the expression of chick Ebf1 and
Ebf3 in the developing neural tube revealed an activation
coincidental with the onset of neurogenesis
(Fig. 1). This was particularly
obvious in the hindbrain, where neurogenesis is known to occur first in
even-numbered rhombomeres (r), and then in odd-numbered ones. A similar
dynamic pattern of activation was seen for Ebf1 and Ebf3,
which were activated throughout the hindbrain except in r3 and r5 at stages
HH13-HH14 (Fig. 1A,B);
expression in these latter rhombomeres occurred only from stage HH15 (data not
shown). Sections through the spinal cord revealed that Ebf1 and
Ebf3 expression was mainly associated with the mantle layer, and was
likely to correspond to differentiating post-mitotic neurones
(Fig. 1C-F). These expression
patterns suggest that Ebf gene activation may constitute a general feature of
neuronal differentiation, in this part of the CNS at least. In addition, they
are very similar to those observed for the murine orthologs
(Garel et al., 1997;
Wang et al., 1997
). The
conservation of gene sequences and expression profiles suggest that Ebf genes
perform similar functions in mammals and birds.
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These experiments indicate that Ngn2 and NeuroM can promote Ebf gene expression during neuronal differentiation. They suggest that proneural and early differentiation genes lie upstream of the Ebf genes in the cascade of events controlling neurogenesis.
Ebf1 misexpression promotes neuronal differentiation
To directly assess Ebf gene function in neurogenesis, we first performed
gain-of-function experiments by using in ovo electroporation in the chick
neural tube with the pAdRSV-Sp expression vector. We chose to misexpress the
mouse Ebf1 gene because the complete coding sequence was available
and this gene appears to be very similar to the chick ortholog, both in terms
of sequence and expression pattern. In the first series of experiments, stage
HH15 embryos were co-electroporated with Ebf1 and lacZ
expression plasmids to identify the electroporated cells [it has been shown
that under these conditions, most electroporated cells receive both plasmids,
(Dubreuil et al., 2000)]. As a
control, some embryos were electroporated with the lacZ expression
plasmid alone. In the control experiments, analysis of the
lacZ-positive cells, performed by X-gal staining, indicated that, as
expected, 20 hours after electroporation most cells presented a morphology and
a localisation of neuroepithelial progenitors
(Fig. 3A). This was still the
case for the majority of the electroporated cells at 40 hours after
electroporation, but at that time some X-gal-positive cells were restricted to
the mantle layer and had presumably undergone differentiation
(Fig. 3B). Analysis of the
distribution of X-gal-positive cells in neural tubes co-electroporated with
the Ebf1 expression plasmid revealed a very different situation:
whereas at 20 hours after electroporation most of the cells presented a
neuroepithelial morphology (Fig.
3C), like in the control, at 40 hours essentially all of the
lacZ-positive cells were localised in the mantle layer
(Fig. 3F). A time course
analysis indicated that the migration of Ebf1-electroporated cells
was largely engaged at 24 hours and was almost completed at 30 hours
(Fig. 3D,E). Therefore ectopic
expression of Ebf1 in neuroepithelial progenitors appears to result
in the early onset of cell migration toward the mantle layer.
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Finally, we investigated whether forced Ebf1 expression would
result in modifications in the expression pattern of neuronal and
neuroepithelial markers. As indicated above, at around stages HH13-HH14 the
hindbrain is an interesting region in which to analyse neuronal
differentiation because, in contrast to even-numbered rhombomeres,
odd-numbered rhombomeres are still largely devoid of differentiated neurones,
as indicated by neurofilament labelling
(Fig. 3K). Analysis of
neurofilament expression in Ebf1-electroporated hindbrain revealed
both an increased number of differentiated neurones in even-numbered
rhombomeres and an early onset of neuronal differentiation in odd-numbered
ones (Fig. 3L). Furthermore,
double-labelling for HA-Ebf1 and neurofilaments or another neurone-specific
marker, neuronal class III ß-tubulin (Tuj1), indicated that most
Ebf1-positive cells also expressed these markers
(Fig. 3M-O). An important
aspect of the neuronal differentiation programme is the shift in the
expression pattern of adhesion molecules that is associated with neuronal
migration: for example, N-cadherin is expressed in neuroepithelial
progenitors, whereas R-cadherin expression is activated later during
neurogenesis and has been suggested to be associated with post-mitotic cells
(Inuzuka et al., 1991b;
Redies and Takeichi, 1996
). We
found that Ebf1 ectopic expression led to the repression of
N-cadherin expression and the activation of R-cadherin expression
(Fig. 3P-R). Therefore, Ebf1
promotes downregulation of a progenitor-associated adhesion molecule and
expression of a neurone-associated one. This may play a role in the early
onset of the migration of the electroporated cells towards the mantle layer.
In conclusion, these data indicate that ectopic expression of Ebf1 in
neuroepithelial progenitors promotes neuronal differentiation, as indicated by
modifications in the expression of several markers.
Ebf1-mediated neurogenesis involves activation of bHLH
genes
As Ebf1 misexpression promotes neurogenesis in a similar way to
proneural genes, we wondered whether this outcome might be reached through
activation of the proneural genes. We therefore tested the effect of
Ebf1 misexpression on Ngn1 and Ngn2, as well as on
the achaete-scute-related Cash1 gene. Ten hours after
electroporation, expression of both Ngn1 and Ngn2 was
induced on the experimental side (Fig.
4A,B). By contrast, Cash1 expression was not affected
(Fig. 4C). At later stages,
consistent with the neuronal differentiation induced by Ebf1, we
observed a general downregulation of the proneural genes. In the case of both
Ngn1 and Cash1, this was already visible at 20 hours after
electroporation, whereas the decrease in Ngn2 expression was only
observed after 30 hours (Fig.
4D-F, and data not shown). These data indicate that Ebf1 can
promote the expression of some, but not all, proneural genes, which suggests
the existence of specific, positive regulatory loops linking these genes.
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Ebf1 misexpression alters neuronal subtype
As Ebf1 is able to promote neuronal differentiation, we wondered whether it
might also interfere with the specification of neuronal identity. To
investigate this possibility, we analysed the effect of Ebf1
misexpression on neuronal subtype characteristics. At the stage of collection
of the embryos (30 hours after electroporation at stage HH15), thoracic spinal
cord neurones are normally distributed between Islet1/Islet2-positive
populations, which correspond to motoneurones located in the ventral region,
and Lim1- and/or CRABPI-positive populations, which
correspond to interneurones, mostly located dorsally to the motoneurone pool
(Tsuchida et al., 1994;
Vaessen et al., 1990
), with no
co-expression of these two types of markers
(Fig. 5A-E,G). Upon
Ebf1 electroporation, we observed an extension of the
CRABPI- and Lim1-positive domains into the motor column
(Fig. 5A-E). Furthermore, these
markers were also detected in cells still located within the ventricular zone;
they are normally restricted to the mantle layer
(Fig. 5A,B). Double-labelling
experiments indicated that CRABPI- or Lim1-positive cells
located at the level of the motor column were negative for Islet1
(Fig. 5C,E). Finally, double
labelling for electroporated Ebf1 and for Islet1/Islet2 revealed that all
electroporated cells were negative for Islet1/Islet2, even though they were
located at the level of the motor column
(Fig. 5G). Performing the
electroporation experiments at different stages of embryo development did not
affect this result (Fig. 5F,H). In all cases, the electroporation of Ebf1 resulted in a reduction of
the pool of Islet1/Islet2-positive cells
(Fig. 5F-I).
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A dominant-negative Ebf protein prevents neuronal
differentiation
The previous analyses have indicated that Ebf genes can interfere at
various levels of the neuronal differentiation pathway. In order to identify
their precise role during normal neurogenesis, we attempted to generate a
dominant-negative molecule. We introduced a deletion in the Ebf1
expression vector, resulting in the elimination of the N-terminal 107 amino
acids of the wild-type protein. Such a deletion retains the dimerisation
domain but has been shown previously to obliterate Ebf1 DNA-binding activity
(Hagman et al., 1993).
Furthermore, a similar construct derived from the Xenopus Ebf2 gene
was shown to have a dominant-negative activity
(Dubois et al., 1998
).
To investigate whether the deleted protein, termed Ebf, possessed a
dominant-negative activity, we performed co-electroporation experiments with
varying amounts of the wild-type Ebf1 expression construct and
examined the effect on neuronal differentiation at the hindbrain level, as
revealed by neurofilament staining (Fig.
6A-D). A concentration of 2 µg/µl of the
Ebf
expression plasmid was able to prevent precocious neuronal differentiation
induced by wild-type Ebf1 at concentrations varying between 1 and 2
µg/µl (Fig. 6B,C, and
data not shown). This suggests that
Ebf antagonises wild-type Ebf1 and,
therefore, can act as a dominant-negative molecule. Furthermore, at lower
concentrations of the Ebf1 expression plasmid, or in its absence,
Ebf led to inhibition of neuronal differentiation
(Fig. 5D, and data not shown).
This observation suggests that Ebf family members are involved in normal
neuronal differentiation, and that
Ebf can also antagonise endogenous
Ebf1 and, presumably, other members of the family owing to heterodimer
formation. To better establish this point, we examined the effect of
electroporation of
Ebf on the expression of the interneurone
and motoneurone markers, CRABPI and Islet1, respectively. In both cases, we
observed a limited but significant decrease in expression on the experimental
side (Fig. 6E,F).
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In conclusion, our data suggest that Ebf activity is necessary for neuronal differentiation, but that it is not required for earlier stages of neurogenesis.
Antagonizing Ebf blocks neuronal differentiation and migration
without interfering with cell cycle exit
As expression of Ebf prevents expression of neuronal
markers, we then investigated whether it could also interfere with neuronal
migration and/or cell cycle exit. For this purpose, we performed
co-electroporation of the
Ebf vector with a construct that
expresses green fluorescent protein (GFP), in order to trace electroporated
cells. To evaluate cell cycle exit, we carried out pulse-labelling with BrdU
as described above.
Ebf-electroporated cells were mostly found
within the ventricular zone, which is in contrast to control cells
electroporated with the mutant Krox20 construct, which were
distributed between the ventricular zone and the mantle layer, and
Ebf1-electroporated cells, which were largely confined to the mantle
layer (Fig. 7A-D). These data
suggest that expression of
Ebf prevents migration towards the
mantle layer. In addition,
Ebf-electroporated cells did not
express the neuronal marker Tuj1 (Fig.
7I), indicating that
Ebf prevented neuronal
differentiation, which is in agreement with the above data
(Fig. 6). Analysis of
BrdU-positive cells among electroporated cells indicated that they represented
similar proportions after electroporation with
Ebf or with the
control construct, whereas, as expected, this proportion was dramatically
reduced after electroporation with Ebf1
(Fig. 7A-D). This indicates
that despite blocking neuronal migration and differentiation,
Ebf did
not affect the proportion of cells in S-phase, and presumably of proliferating
cells. Accordingly, the proportion of electroporated BrdU-negative cells
within the ventricular zone after electroporation with
Ebf was
much higher than in the control, suggesting that cells had exited the cell
cycle but stayed within the ventricular zone
(Fig. 7D).
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As expression of Ebf appears to block neuronal
differentiation of cells engaged in the neurogenic pathway, we wondered about
the effects of this contentious situation on cell fate and survival at later
stages. For this purpose, we analysed cell apoptosis by TUNEL assay, 40 hours
after co-electroporation of the neural tube with either the
Ebf expression construct or the mutant Krox20
expression plasmid as a control, together with a lacZ vector as a
tracer. We found that in the control case some apoptosis is observed
specifically in the electroporated side
(Fig. 7L). This phenomenon
might be related to non-specific effects of high levels of expression of the
electroporated genes. Cells electroporated with
Ebf were still
largely located within the ventricular zone (89±3%). Electroporation of
Ebf led to a significant increase of the number of
TUNEL-positive cells (3.5±1-fold) in the electroporated side, as
compared with the control case (Fig.
7L,M, note that at least half of the apoptotic cells in the
experimental side express lacZ, although this is not visible in the
figure because of the very low level of ß-galactosidase, possibly due to
apoptosis). These data suggest that the
Ebf-mediated block in neuronal
differentiation can lead to cell death in at least a subset of the affected
cells.
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Discussion |
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The Ebf genes in the neurogenic cascade
Our conclusions on Ebf gene regulation and function are detailed below, and
are based on the following observations.
(1) Analysis of Ebf1 and Ebf3 mRNAs in the chick neural
tube indicated that their accumulation is coincidental with the onset of
neurogenesis and that they are detected within the entire mantle layer
(Fig. 1). This is in agreement
with the expression pattern of the mouse orthologs
(Garel et al., 1997;
Wang et al., 1997
), and shows
that these genes are expressed at a high level in early post-mitotic neurones
and that their expression is maintained during neuronal differentiation. Low
level, scattered expression has also been observed in the neuroepithelium for
mouse Ebf2 and Ebf3, presumably corresponding to cells en
route to the mantle layer (Garel et al.,
1997
; Pattyn et al.,
2000
).
(2) Forced expression of both Ngn2, a proneural gene, and NeuroM, an early neuronal differentiation regulator, promoted Ebf1 and Ebf3 expression (Fig. 2), indicating that the latter genes are downstream of the former in the neurogenic cascade, consistent with Ebf gene expression pattern.
(3) Expression of a dominant-negative molecule, which presumably antagonizes all Ebf activities, did not affect cell cycle exit, but prevented neuroepithelial precursor migration towards the mantle layer and expression of differentiation markers (Figs 6, 7). Furthermore, the dominant-negative Ebf was also able to prevent neuronal differentiation and migration induced by the forced expression of Ngn2, but it did not affect the endogenous expression of this latter gene. Together, these observations suggest that Ebf genes play an essential role in cell engagement into neuronal differentiation and migration towards the mantle layer, coupling these processes to cell cycle exit.
(4) In agreement with a role of Ebf genes in the control of neuronal differentiation and migration, misexpression of Ebf1 in neuroepithelial progenitors promoted these processes (Fig. 3), which indicates that Ebf genes are both necessary and sufficient. However, surprisingly, forced expression of Ebf1 also led to exit from the cell cycle. This was correlated with a transient reinforcement of Ngn1 and Ngn2 (Fig. 5), and NeuroM (data not shown), expression. We could actually demonstrate that induction of the complete neurogenic programme by Ebf1 was largely dependent on bHLH proteins, presumably including proneural gene products, as shown by its inhibition by the bHLH antagonist Id2 (Fig. 3S,T). At this stage, we cannot exclude that forced high level expression of Ebf1 in neuroepithelial progenitors may lead to non-physiological proneural gene activation, subsequently resulting in the activation of the complete programme. An alternative explanation, involving a second function of Ebf genes, can nevertheless be envisaged and is discussed below.
(5) We have shown that Ebf1 misexpression also leads to changes in the balance of neuronal subtypes (Fig. 5). This suggests the existence of a third level of intervention of Ebf genes in the neurogenic cascade.
On the basis of these different observations, we propose to position the Ebf genes in the neurogenic cascade in the spinal cord and the hindbrain as indicated in Fig. 8. According to this model Ebf genes are downstream to proneural genes and cell cycle exit, but are absolutely required for neuronal differentiation and migration towards the mantle layer. The details of our conclusions are discussed below.
|
However, uncoupling cell cycle exit from neuronal differentiation and
migration may not be without consequences. Our analysis of cell death
following expression of dominant-negative Ebf indicates that this
treatment increases the proportion of apoptotic cells among electroporated
cells (Fig. 7), which suggests
that the cells might be able to sense this abnormal uncoupling and,
consequently, enter into a cell death programme. A related interpretation is
that the cells could simply sense the block in differentiation when they are
already too far engaged in the commitment process. In several systems, a block
in cell differentiation is accompanied by cell death, and this is also the
case in the developing striatum of Ebf1 null mice
(Garel et al., 1999).
As Ebf expression is required for both neuronal differentiation and
migration towards the mantle layer, a subsequent question is whether Ebf genes
independently contribute to each of these two manifestations, or whether
differentiation is a consequence of migration or vice-versa. In favour of a
direct role of Ebf genes in migration, we have observed that Ebf1
ectopic expression results in a modification of the pattern of expression of
adhesion molecules, with repression of N-cadherin and induction of R-cadherin.
Such modifications have been proposed to be involved either in the release of
undifferentiated progenitors from the ventricular zone and/or in the promotion
of their migration towards the mantle layer
(Redies and Takeichi, 1996).
Furthermore, additional evidence suggests a role of Ebf genes in the control
of cellular adhesion, and of cellular and axonal migration
(Prasad et al., 1998
;
Garel et al., 1999
;
Garel et al., 2000
;
Corradi et al., 2003
).
Nevertheless, our work suggests that Ebf genes also control aspects of
neuronal differentiation independently from the migration towards the mantle
layer. Neuronal markers like Lim1 and CRABPI, which are
normally restricted to the mantle layer, are expressed in
Ebf1-electroporated cells while they are still within the ventricular
zone. In addition, in the striatum primordium of Ebf1 null-mutant
embryos, cells in the mantle layer show an aberrant differentiation pattern,
being unable to downregulate genes normally restricted to the SVZ, or to
activate some mantle-specific genes (Garel
et al., 1999
). Together these data suggest that the function of
Ebf genes is not likely to be restricted either to the promotion of migration
towards the mantle layer or to the induction of neuronal differentiation, but
rather that these genes directly contribute to both types of processes, and
may actually coordinate them.
Additional, putative functions of Ebf genes in neuronal commitment
and subtype specification
As indicated above, we do not know whether the transient activation of
Ngn2 and NeuroM by Ebf1
(Fig. 4) is physiological, in
particular because expression of the dominant-negative Ebf does not
seem to affect the expression of these genes
(Fig. 6). However the
establishment of positive-feedback loops, involving proneural, Ebf and
possibly other genes, might be involved in the stabilisation of the committed
state among selected progenitors (Fig.
8). Another possibility is that Ebf genes might mediate a signal
acting after cell cycle exit and ensuring that differentiating cells cannot
resume proliferation, as has been proposed for NeuroD
(Mutoh et al., 1998). If there
is redundancy in the establishment or maintenance of these loops, the latter
might not be revealed by the loss-of-function mediated by the
dominant-negative Ebf. A similar feedback mechanism involving
Xcoe2/Ebf2 has been proposed during primary neurogenesis in
Xenopus (Dubois et al.,
1998
). This raises the possibility that different aspects of the
functions of Ebf genes, and of their relationships with other genes involved
in the control of neurogenesis, have been conserved during vertebrate
evolution. By contrast, Xenopus Ebf3 is expressed later than
Xcoe2, is not involved in maintaining XNgn1 expression and
has been proposed to be required only in late neuronal differentiation,
downstream of XNeuroD (Pozzoli et
al., 2001
). Although our experiments did not specifically address
the precise level of action of each Ebf gene, the expression data (this work)
(Garel et al., 1997
;
Malgaretti et al., 1997
;
Wang et al., 1997
;
Pattyn et al., 2000
), together
with the absence of a general neuronal phenotype associated with the
Ebf2 null mutation (Corradi et
al., 2003
), are consistent with an early role of Ebf1 and
Ebf3, coincidental and redundant with that of Ebf2.
Therefore, Ebf2 and Ebf3 have functionally diverged in
Xenopus, whereas they may have conserved similar early neurogenic
function in higher vertebrates.
Combinatorial expression of homeobox genes in neural progenitors,
established according to their DV location and in response, in particular, to
sonic hedgehog signalling, has been shown to play an essential role in
neuronal subtype specification in the spinal cord
(Jessell, 2000). Proneural
genes also have restricted patterns of expression along the DV axis and
recently they have been implicated in the specification of neuronal subtype as
well (Fode et al., 2000
;
Gowan et al., 2001
). In this
study, we have shown that Ebf1 misexpression in spinal cord
progenitors leads to repression of motoneurone markers (Islet1/Islet2) and
activation of interneurone markers (Lim1 and CRABPI), at the level of the
motor column (Fig. 5). This
suggests that progenitors normally fated to become motoneurones according to
their DV location are reprogrammed towards an interneurone fate. This was
unexpected as Ebf genes are normally expressed in differentiating motoneurones
(Fig. 1) (Garel et al., 1997
). We can
provide two possible, and non-exclusive, explanations for our observations.
Firstly, it is possible that high levels or inappropriate timing (leading to
premature cell cycle exit in particular) of Ebf1 expression in
neuroepithelial progenitors leads to modifications in the combinatorial
expression of genes involved in early DV specification (e.g. homeobox genes,
proneural genes) and, consequently, but indirectly, to fate changes. Indeed,
we have shown that Ebf1 misexpression has different effects on the
expression of three proneural genes, promoting the expression of Ngn1
and Ngn2, but not of Cash1
(Fig. 4). Secondly, because the
Ebf genes appear as major regulators of neuronal differentiation, it is
possible that they directly control the expression of neuronal
subtype-specific genes. Indeed, we have shown that Ebf1 inactivation
in the striatum primordium prevents the activation of CRABPI
(Garel et al., 1999
), and that
forced expression of Ebf1 leads to ectopic activation of
CRABPI not only in the motor column but also in the ventricular zone
(Fig. 5). It is therefore
possible that higher than normal levels of Ebf expression in differentiating
neurones directly alters the balance between subtype specification genes.
Further analyses will be required to precisely delineate the possible function
of Ebf genes in this aspect of neuronal differentiation.
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ACKNOWLEDGMENTS |
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