1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, BP 163, 67404 Illkirch, Cedex, CU de Strasbourg, France
2 Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell
Biology, Am Fassberg 11, D-37077 Göttingen, Germany
* Present address: Institute of Cell Biology and Tissue Engineering, Via di
Castel Romano 100/102, 00128 Rome, Italy
Present address: Division of Molecular Neurobiology, National Institute for
Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Author for correspondence (e-mail:
fguille{at}nimr.mrc.ac.uk)
Accepted 9 April 2003
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SUMMARY |
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Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.
Key words: Spinal cord, Cerebral cortex, Transcriptional enhancer, Chick, Electroporation
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INTRODUCTION |
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Pax6 is a HD protein involved in the establishment of progenitor domains in
the ventral neural tube and in the specification of progenitors to particular
cell fates. Pax6 expression is repressed by Shh signalling, resulting in a
ventrallow-medialhigh gradient of Pax6 protein in the
spinal cord and its exclusion from ventral-most progenitors
(Ericson et al., 1997).
Subsequently, cross-regulatory interactions between Pax6 and the HD
gene Nkx2.2 sharpen the boundary between the Nkx2.2-positive, V3
interneuron progenitor domain adjacent to the floor plate and the neighboring
Pax6low motor neuron progenitor domain
(Briscoe et al., 2000
).
Analysis of mouse and rat embryos homozygous for the naturally occurring null
mutation in the Pax6 gene, Small eye (sey), has
revealed that Pax6 is required for the generation of the V1 and V2
subtypes of ventral interneurons and the correct specification of subsets of
spinal and hindbrain motor neurons
(Ericson et al., 1997
;
Takahashi and Osumi, 2002
).
However, no specific function has yet been ascribed to the distinct
concentration gradient of Pax6 protein in the ventral spinal cord.
Strikingly similar regulatory interactions between HD genes are responsible
for partitioning the telencephalic primordium into distinct territories
(reviewed by Wilson and Rubenstein,
2000). Pax6 is also expressed in a graded manner in the dorsal
telencephalon, reaching highest levels in a lateral and caudal domain of the
cerebral cortex and gradually diminishing towards the medial-rostral cortex.
Cross-regulatory interactions between Pax6 and the HD gene
Gsh2 have been shown to establish the border between the cerebral
cortex, dorsally, and the lateral ganglionic eminence, ventrally
(Toresson et al., 2000
;
Yun et al., 2001
). Pax6 has
also been shown to control many properties of cortical cells, including the
proliferation of cortical progenitors, their neuronal commitment, and the
migration of newborn neurons (e.g.
Stoykova et al., 2000
;
Muzio et al., 2002
;
Heins et al., 2002
;
Estivill-Torrus et al., 2002
).
Its graded expression has been implicated in the regionalization of the
neocortex into distinct areas (Bishop et
al., 2000
).
The proneural genes that encode basic helix-loop-helix (bHLH) transcription
factors, also play an important role in establishing the fates of neural
progenitors (Kageyama and Nakanishi,
1997; Bertrand et al.,
2002
). Members of this gene family, which include Mash1, Math1 and
the neurogenins, have the dual function of promoting the differentiation of
individual progenitors, and of selecting the particular neuronal or glial
lineage along which progenitors differentiate. In the spinal cord, the
neurogenin gene Ngn2 has been shown to promote cell cycle arrest and
neuronal differentiation of neuroepithelial cells
(Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Scardigli et al., 2001
).
Ngn2 has also been shown to contribute to the specification of motor
neuron progenitors, acting in conjunction with a major determinant of motor
neuron fate, the bHLH protein Olig2
(Mizuguchi et al., 2001
;
Novitch et al., 2001
). In the
telencephalon, Ngns have similar roles in neuronal commitment and
specification of the identity of cortical progenitors
(Fode et al., 2000
;
Nieto et al., 2001
;
Sun et al., 2001
).
Proneural proteins are, like HD proteins, expressed in restricted
progenitor domains, and cross-repressive interactions are similarly involved
in establishing the sharp dorsoventral borders that separate these domains
(Fode et al., 2000;
Gowan et al., 2001
) (reviewed
in Bertrand et al., 2002
). In
the spinal cord, Ngn3 is expressed in a ventral domain immediately
adjacent to the floor plate, and Ngn1 and Ngn2 are expressed
throughout most of the basal plate and in restricted domains of the alar
plate, while Mash1 is expressed in a large part of the alar plate and
Math1 is expressed in a dorsal domain immediately adjacent to the
roof plate. In the telencephalon, Mash1 is expressed at high levels
in ventral progenitors and at reduced levels in a subset of dorsal
progenitors, contrasting with the restricted expression of Ngns in
dorsal progenitors. In addition to this strict spatial regulation, there is
recent evidence that the precise timing of proneural gene expression is
important for the correct specification of progenitors. For example, it has
been proposed that down-regulation of Ngn2 expression in the motor
neuron progenitor domain is involved in the transition from motor neuron to
oligodendrocyte generation in this region of the spinal cord
(Zhou et al., 2001a
).
Despite the importance of these proneural expression patterns for the
diversification of progenitor populations, little is known of how they are
established. The restriction of proneural gene expression along the
dorsoventral axis of the neural tube suggests an implication of inductive
signals produced by dorsal and ventral organizing centers. Indeed, there is
evidence that BMP signals simultaneously regulate the expression of proneural
and HD proteins in the dorsal spinal cord
(Timmer et al., 2002), and
that Shh induces Mash1 expression in the ventral telencephalon
(Yung et al., 2002
). In the
ventral spinal cord, several factors, which are themselves regulated by Shh
signalling, have been shown to control the expression of Ngn genes.
Nkx2.2 is required for the expression of Ngn3 in a domain
adjacent to the floor plate (Briscoe et
al., 1999
), and Olig2 regulates Ngn2 expression in
progenitors of motor neurons (Mizugushi et al., 2001;
Novitch et al., 2001
;
Zhou and Anderson, 2002
). In
the cerebral cortex, Ngn2 has been shown to be regulated by Pax6
(Stoykova et al., 2000
;
Toresson et al., 2000
;
Yun et al., 2001
). Thus,
regulatory interactions between patterning genes and proneural genes may be
involved in coordinating the distinct genetic programs underlying the regional
specification of progenitors and their lineage commitment.
To further elucidate the mechanisms controlling the spatial and temporal
expression of Ngn2, we have initiated a study of the regulatory
sequences of this gene and identified four distinct enhancer elements
(Scardigli et al., 2001).
These enhancers drive gene expression in subsets of the Ngn2
expression domain, and together cover most of this domain. Interestingly,
analysis of Ngn2 enhancers in small eye mice revealed that the
activity in the ventral spinal cord of one of the enhancers, named E1,
requires Pax6 function, probably explaining the role of Pax6 in
regulating Ngn2 expression in this domain
(Scardigli et al., 2001
). In
contrast, Pax6 only has a minor role in the regulation of other Ngn2
enhancers, thus explaining that much of Ngn2 expression in the spinal
cord is unaffected in Pax6 mutants. In this work, we have further
characterized the regulation of the E1 element by Pax6. We have specifically
asked whether this interaction is direct, and whether Pax6 controls the
spatial domain of activity of this Ngn2 enhancer.
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MATERIALS AND METHODS |
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To generate the constructs
4xE1.1ßglobinlacZ and
4xE3.2ßglobinlacZ, 26 bp-long
oligonucleotides corresponding to the E1.1 and E3.2 binding sites, with a
BamHI restriction site and a BglII site at either end, were
hybridized, oligomerized, and cloned into the pKSBluescript vector
(Stratagene). Inserts containing 4 copies of the oligonucleotide were selected
and cloned as Not1-Spe1 fragments into the
ßglobinlacZ (BGZA) vector
(Yee and Rigby, 1993).
Generation, genotyping and analysis of transgenic and mutant
mice
Transgenic mice were generated by standard procedures using fertilized eggs
from FVBN mice, and founder animals were genotyped for the lacZ
sequence by PCR as previously described
(Scardigli et al., 2001).
PAX6YAC transgenic mice [(Schedl
et al., 1996
) kindly provided by A. Schedl] were bred with
E1hsplacZ transgenic mice. PAX6YAC transgenic embryos were
identified by their eye phenotype, and E1hsplacZ embryos by X-gal
staining Embryos were dissected from the uterus in cold PBS and fixed at room
temperature in 4% paraformaldehyde for 30 minutes to 1 hour depending on the
stage. Whole-mount X-gal staining was performed as described
(Beddington et al., 1989
).
After staining, some embryos were embedded in 1-2% agarose and
vibratome-sectioned at 100 µm.
In ovo chick electroporation
In ovo electroporation of chick embryos was performed as described
previously (Funahashi et al.,
1999) using a BTX electroporator (Electro Square Porator, ECM
830), with the following parameters: 3 times 25 V square pulses of 30
mseconds. DNA was purified using a Maxiprep EndoFree kit (Qiagen) and injected
into the neural tube of HH stage 10-12 (E1.5) embryos
(Hamburger and Hamilton,
1992
). 1-1.5 µl of reporter construct at a concentration of 2
µg/µl was injected, together with 0.2 µg of CMVeGFP plasmid
(Clontech) as tracer, and in some experiments the same amount of
CMVPax6 construct (Marquardt et
al., 2001
). Either 6 hours or 48 hours after electroporation,
GFP-positive embryos, identified with UV light under a dissection microscope
(Leica MZFL3), were collected and analyzed by immunocytochemistry. At least
four electroporated embryos were analysed in each experiment.
Immunohistochemistry
Mouse and chicken embryos were fixed in 4% paraformaldehyde at room
temperature for 30 minutes to 2 hours depending on the stage, impregnated with
20% sucrose overnight, embedded in OCT compound (Tissue Tek), and
cryosectioned at 10 µm. Double immunofluorescence experiments were
performed as previously described
(Scardigli et al., 2001) by
simultaneous incubation with two primary antibodies. The following antibodies
were used: mouse monoclonal anti-ß-galactosidase (Promega), rabbit
polyclonal anti-ß-galactosidase (5 prime-3 prime, Inc.), rabbit
polyclonal anti-GFP (Molecular Probes), rabbit polyclonal anti-Pax6 (Babco),
mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank), rabbit
polyclonal anti-cNgn2 [(Zhou et al., 2001) kindly provided by D. Anderson],
rabbit polyclonal anti-Nkx6.1 and guinea pig polyclonal anti-Nkx6.2
[(Vallstedt et al., 2002) kindly provided by J. Ericson]. Alexa 488- and Alexa
594-coupled secondary antibodies were purchased from Molecular Probes.
Whole-mount immunocytochemistry was performed on HH stage 13-15 chicken
embryos collected in phosphate-buffered saline (PBS), fixed in 4%
paraformaldehyde at room temperature for 30 minutes, washed in PBS, and
incubated overnight at 4°C with the appropriate antibody diluted in 0.1%
Triton X-100, 3% bovine serum albumin and 10% fetal calf serum in PBS. Embryos
were then extensively washed in PBS plus 0.1% Tween 20 and incubated overnight
at 4°C with a secondary antibody. Embryos were then washed and flat
mounted in AquaPolymount (Polysciences Inc.). Sections and whole-mount samples
were analysed using a confocal microscope (Leica Sp1). 3 stacks of pictures
were merged to generate images of sections and 10 stacks of pictures
(representing 25 µm in thickness) were merged to generate images of
whole-mount embryos.
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RESULTS |
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To determine if the regulation of E1 by exogenous Pax6 reflects a similar
regulation of the endogenous Ngn2 gene, the CMVPax6
construct was co-electroporated with a GFP expression construct into the
neural tube of HH stage 10-12 embryos, and Ngn2 expression was examined 6
hours later by immunocytochemistry. Increased level of Pax6 protein in
electroporated cells correlated with a strong expression of Ngn2, which was
not observed in the non-electroporated side
(Fig. 2A,B), thus indicating
that exogenous Pax6 protein is able to induce endogenous Ngn2 expression. Pax6
is thus a limiting factor for the activity of E1 as well as for the expression
of endogenous Ngn2 in the early neural tube. Altogether, loss-of-function
experiments (Fig. 2A-D)
(Scardigli et al., 2001) and
gain-of-function experiments (Fig.
2E-J) demonstrate that Pax6 is both necessary and
sufficient to activate the E1 enhancer and induce Ngn2 expression in
the embryonic neural tube. We next examined the molecular mechanisms
underlying the regulation of E1 by Pax6.
A low affinity Pax6 binding site is present in the E1 enhancer
To determine if the regulation of Ngn2 expression by Pax6 is
direct or indirect, we searched for the presence of putative Pax6 binding
sites. A sequence with high similarity to published consensus binding
sequences was found in the E1 element. This sequence, designated E1.1,
contains 11 nucleotides of the 16-nucleotide consensus binding sequence for
the paired box of Pax6 (Epstein et al.,
1994; Czerny and Busslinger,
1995
). Putative Pax6 binding sites were also found in other
Ngn2 enhancers (Fig.
3A and data not shown). Ngn2 enhancer elements contain
blocks of sequence that are highly conserved between the mouse and human
Ngn2 genes (Scardigli et al.,
2001
). The E1 element has one block of 544 bp, situated between
residues 63 and 607, that is 94% identical between the two species
(Fig. 3A). The E1.1 sequence is
located between residues 583 and 600, at the 3' end of this block of
homology, and 14/16 bp are conserved in the human sequence
(Fig. 3A).
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The above data indicate that the E1.1 sequence bind Pax6 in vitro, but with
relatively low affinity. To determine if the E1.1 sequence can bind
Pax6 protein in vivo and recruit it at a promoter, a concatemer of 4 copies of
E1.1 was cloned in front of the basal ßglobin promoter driving
expression of lacZ (Yee and
Rigby, 1993). The capacity of this construct, designated
4xE1.1ßglobinlacZ, to recruit Pax6 and
activate the ßglobin promoter, was compared with that of a
similar construct containing a concatemer of 4 copies of the E3.2 sequence
(4xE3.2ßglobinlacZ). These two
constructs were electroporated into the neural tube of HH stage 10-12 chicken
embryos, which were tested for ßgal expression 6 hours later. While the
4xE3.2ßglobinlacZ construct was
efficiently activated in a large number of cells, the
4xE1.1ßglobinlacZ construct drove
ßgal expression in only very few cells
(Fig. 3Ca,c), suggesting that
4xE3.2ßglobinlacZ, and not
4xE1.1ßglobinlacZ, can be activated by
the low levels of Pax6 protein present in the early neural tube. To determine
whether 4xE1.1ßglobinlacZ can be
activated by higher concentrations of Pax6, the construct was
co-electroporated with a CMVPax6 expression vector. A significant
increase in the number of ßgal-positive cells was observed in this
experiment when compared with the electroporation of
4xE1.1ßglobinlacZ alone (compare
Fig. 3Ce with 3Ca), indicating
that this construct can be activated by high concentrations of Pax6 protein.
In contrast, the activity of the
4xE3.2ßglobinlacZ construct was not
significantly enhanced when co-electroporated with CMVPax6. Together,
this data indicates that the E1.1 sequence corresponds to a low affinity
binding site for Pax6, while E3.2 is a site with higher affinity, thus
confirming the results obtained in vitro
(Fig. 3B).
The Pax6 binding site is required for the activity of the E1
element
The activity of the E1 element is known to be dependent on Pax6
(Scardigli et al., 2001)
(Fig. 2) and we have identified
a Pax6 binding site in E1 (Fig.
3), which suggests that occupation of this site by Pax6 may be
important for E1 activation. To test this possibility, we disrupted the E1.1
site and tested the effect of this mutation on E1 activity in transgenic mice.
Nucleotide substitutions were introduced into the core sequence of the
Pax6-binding site (see Materials and Methods), and the mutated E1 element
(mtE1) was inserted in front of the basal hsp68 promoter driving
expression of lacZ. In E10.5 embryos carrying the
mtE1hsplacZ transgene, ß-gal activity was greatly diminished in
the neural tube, in comparison with embryos carrying a wild-type E1 construct
(compare Fig. 4B,D with 4A,C;
n=4). However, mutation of the E1.1 sequence did not completely
abolish E1 activity, even though E1 activity is completely dependent on
Pax6 function (Scardigli et al.,
2001
). The residual activity of mtE1 in the neural tube was
observed at a dorsoventral position similar to that of wild-type E1
(Fig. 4D) (Scardigli et al., 2001
). In
contrast, mtE1 was completely inactive in the telencephalon, similar to what
is observed with wild-type E1 in the absence of Pax6
(Fig. 4B). Together, these
results demonstrate that the Pax6 binding site present in E1 has an essential
role in governing the activity of the element in both spinal cord and
telencephalon.
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The modified E1 element containing an optimized E1.1 sequence (consE1) was cloned in a ßglobinlacZ vector to test its activity in chicken and mouse embryos. Constructs were electroporated into the neural tube of HH stage 10-12 chicken embryos, and first analyzed 6 hours later. As shown earlier, wild-type E1 drives ß-gal expression in only a few cells at this stage because of the low endogenous level of Pax6 expression (Fig. 2G, Fig. 7A). The consE1ßglobinlacZ construct was active in a larger number of cells (Fig. 7C). This result suggests that introduction of a high affinity Pax6 binding sequence into the E1.1 site results in efficient activation of the E1 element by the low level of Pax6 protein present in the early neural tube. To determine if a consensus Pax6 binding sequence could modify the activity of the E1 element, irrespective of where it was placed in the enhancer, we introduced this sequence into a different site (named E1.2) in the E1 element. The resulting mutated E1 element (named consE1.2) was cloned in the ßglobinlacZ vector and its activity tested. The E1.2 site was chosen because, like E1.1, it contains a sequence with high similarity to the consensus Pax6 binding sequence. However, in contrast to E1.1, mutation of this sequence did not affect the overall activity of the E1 element (data not shown). The mutated consE1.2 element had very low activity in neural tubes harvested 6 hours after electroporation, similar to the wild-type E1 element (n=6; data not shown). This result suggests that the consensus Pax6 binding sequence must be inserted in an active Pax6 binding site in order to modify the response of the E1 element to Pax6.
|
We then examined whether the size of the E1 domain in the cerebral cortex
is controlled by a similar mechanism. For this purpose, transgenic mouse
embryos were generated with the E1ßglobinlacZ and
consE1ßglobinlacZ constructs, and harvested at E11.5
and E12.5 to examine ß-gal activity in the telencephalon. The domain of
ß-gal expression, which was restricted to the lateral cortex in all
E1ßglobinlacZ embryos examined (n=11;
Fig. 8A,C,E) (see also
Scardigli et al., 2001), was
clearly expanded to the dorsolateral cortex in the majority of the embryos
that carried the consE1ßglobinlacZ constructs (2 out of
5 embryos examined at E11.5, and 4 out 6 embryos examined at E12.5;
Fig. 8B,D,F). In the remaining
embryos, the domain of ß-gal expression was the same as in embryos
carrying the control transgene (data not shown). Thus, as demonstrated in the
chicken spinal cord, the presence of a high affinity Pax6 binding sequence at
the E1.1 site results in activation of the E1 element in regions of the
cerebral cortex where Pax6 concentrations are low. This indicates that the
borders of E1 domain in the telencephalon are determined by the affinity of a
Pax6 binding site.
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DISCUSSION |
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Direct regulation of the E1 enhancer by Pax6 involves binding to a
single canonical site
Several studies have recently shown that Pax6 is a regulator of
Ngn2 expression in the presumptive cerebral cortex, and that
activation of Ngn2 is an important mechanism by which Pax6 specifies
the dorsal fate of this territory (Fode et
al., 2000; Stoykova et al.,
2000
; Toresson et al.,
2000
). We have extended these observations to another part of the
embryonic CNS, the spinal cord, where we demonstrate that Pax6 is required for
Ngn2 expression in the p1 and p2 ventral progenitor domains
(Fig. 2)
(Scardigli et al., 2001
). We
provide several arguments supporting the idea that Pax6 regulates
Ngn2 expression in these two territories by directly binding to one
of its enhancers, E1. First, the E1 element loses its activity in the lateral
cortex and ventral spinal cord in a Pax6 null mutant background, and
reciprocally, forced expression of Pax6 in the neural tube leads to activation
of E1 at ectopic locations within 6 hours of overexpression (Figs
2,
5) (see also
Scardigli et al., 2001
).
Second, there is a single canonical and evolutionary conserved Pax6 binding
site in the sequence of E1, and this site (named E1.1) binds Pax6 both in
vitro and in neuroepithelial cells (Fig.
3). Finally, the specific disruption of E1.1 dramatically reduces
the activity of E1 in the spinal cord and eliminates it altogether in the
telencephalon (Fig. 4).
These data provide strong evidence that the interaction of Pax6 with its
cognate binding site E1.1 is important for the activity of E1, but they do not
exclude the possibility that additional mechanisms are involved. A second
canonical Pax6 binding site (E1.2) is indeed present in the E1 sequence, but
this site is unlikely to have a significant role in E1 activation, as it is
not conserved in the human Pax6 locus, and its disruption does not affect the
activity of E1 in the spinal cord or telencephalon (data not shown). However,
E1 conserves a residual activity in the spinal cord when E1.1 is mutated, and
this residual activity must also be Pax6 dependent since E1 is completely
inactive in the ventral neural tube of Sey mutant embryos
(Scardigli et al., 2001).
Thus, Pax6 can weakly activate E1 without interacting with the E1.1 sequence,
suggesting either that it binds weakly to non-canonical sequences in E1, as
reported for the
-cristallin gene
(Kamachi et al., 2001
), or
that it can regulate E1 without directly binding DNA, possibly through
interactions with other DNA binding factors.
There are multiple examples of Pax6 target genes that require synergistic
interactions between Pax6 and co-factors in order to be efficiently
transcribed (Simpson and Price,
2002). We do not know whether this is also the case for the
regulation of the E1 element, but the very high conservation between mouse and
man of a large block of sequence in which the Pax6 binding site is embedded
(504 conserved nucleotides out of 534, see
Fig. 3) strongly suggests that
the activity of E1 involves binding of many factors other than Pax6. We have
also provided evidence, from the comparison of the activity of the E1.1 and
E1.2 sites, that the context of the Pax6 binding site in E1.1 is important. In
particular, the activity of E1 can be increased or reduced by changing the
affinity of the E1.1 sequence for Pax6, whereas similar manipulations of the
E1.2 sequence have little or no impact on E1 activity (data not shown). Thus,
the fact that the E1.1 site has an important role in E1 activity, and E1.2
does not, is not because E1.2 has a lower ability to recruit Pax6 to the
enhancer. More likely, this reflects differences in the environment of E1.1
and E1.2 sequence such as the proximity to binding sites for co-factors with
which Pax6 must interact to activate E1.
The same mechanism controls E1 activity in the spinal cord and
telencephalon
A striking finding of this study is that the same mechanism is employed to
control the expression of Ngn2 in progenitor domains located in two
distant regions of the embryonic CNS, the ventral spinal cord and the dorsal
telencephalon. Similarities in the molecular mechanisms that pattern the
spinal cord and telencephalon along their dorsoventral axis have been noted
before, and include common inductive signals such as Sonic Hedgehog and bone
morphogenetic proteins, related intrinsic determinants, including HD proteins
of the Pax and Nkx families, and bHLH proteins of the Mash and Ngn families,
and in particular the establishment by Pax6 of boundaries between adjacent
progenitor domains, through cross-regulatory interactions with the HD proteins
Nkx2.2 in the spinal cord, and Nkx2.1 and Gsh2 in the telencephalon
(Wilson and Rubenstein, 2000;
Briscoe and Ericson, 2001
;
Schuurmans and Guillemot,
2002
). The activity of E1 in both spinal cord and telencephalon
thus probably reflects a common role of Pax6 in these two territories. It must
be noted however, that E1 is not active in all domains of high Pax6 expression
[e.g. the retina) (Marquardt et al.,
2001
)], suggesting that regional determinants may act as
co-factors, as discussed above, to constrain Pax6 function and restrict E1
activity along the anteroposterior axis of the neural tube.
We have also observed differences in how E1 is regulated in the spinal cord
and telencephalon that are worth noting. In particular, both the introduction
of a high affinity Pax6 binding sequence into the E1.1 site, and the analysis
of E1 activity in the presence of increased dosage of Pax6, resulted in
ectopic activation of E1 in the telencephalon, but not the spinal cord of
transgenic mice. One explanation could be that the concentration gradients of
Pax6 are different in these two territories, with a steeper Pax6 gradient in
the spinal cord possibly limiting the expansion of E1 activity even with a
modified element that responds to lower concentrations of Pax6. Alternatively,
the increased Pax6 gene dosage in PAX6YAC mice, which carry 5 to 7
copies of the entire human Pax6 locus
(Schedl et al., 1996), could
be sufficient to modify the concentration gradient of Pax6 in the cerebral
cortex but not in the spinal cord, if different mechanisms controlling Pax6
expression levels operate in the two territories.
A direct regulatory link between neural patterning and
neurogenesis
The generation of neurons by progenitors in the embryonic nervous system
involves two distinct processes: the commitment of multipotent progenitors to
a neuronal fate, resulting in their differentiation into neurons, and the
specification of progenitors identity, resulting in the differentiation of
neurons of a particular subtype. A number of studies suggest that these two
processes are coupled at several levels. First, proneural bHLH genes, the
major regulators of neuronal commitment in multipotent progenitors, are also
involved in the specification of neuronal identity
(Anderson, 1999;
Brunet and Ghysen, 1999
;
Bertrand et al., 2002
). In
particular, proneural genes have been shown to control some aspects of the
neuronal phenotype, such as the neurotransmission profile, through the
regulation of downstream HD genes that directly activate genes encoding
biosynthetic enzymes for neurotransmitters
(Hirsch et al., 1998
;
Lo et al., 1998
;
Parras et al., 2002
). Second,
the regulation of the proneural genes themselves appears to be intimately
linked with the regionalization of the neural tube, as these genes are
expressed in restricted neuroepithelial domains with well-defined dorsoventral
borders. Some of the genes that are involved in partitioning the
neuroepithelium in dorsoventral progenitor domains have recently been shown to
control the expression of proneural genes in these territories. For example,
the HD protein Phox2b acts as a patterning gene to specify the identity of
branchiomotor neuron progenitors in the hindbrain, and it simultaneously
promotes the neuronal differentiation of these progenitors by upregulating the
expression of the proneural genes Ngn2 and Mash1
(Dubreuil et al., 2002
). A
control of proneural gene expression by neural patterning genes has also been
reported in Drosophila (e.g.
Calleja et al., 2002
). It is
likely to be a general feature of neural development in both invertebrates and
vertebrates.
This work provides the first demonstration that a proneural gene is
directly regulated by a patterning gene in vertebrates, suggesting that neural
patterning and neurogenesis may generally be tightly linked. It is likely that
multiple patterning genes are involved in the generation of the complex
expression patterns of proneural genes. Indeed, Pax6 is essential for the
regulation of only one of the four known enhancer elements of Ngn2
(Scardigli et al., 2001).
Recent work suggests that in Drosophila, regulators of proneural
genes act hierarchically rather than in a combinatorial manner, so that the
number of direct transcriptional activators is actually very small
(Calleja et al., 2002
). Further
studies are necessary to determine whether this holds true for vertebrate
proneural genes.
The role of a Pax6 concentration gradient in the regulation of
Ngn2
Our results demonstrate that the E1 element is regulated by high levels of
Pax6 protein. This element is only active in domains of the spinal cord and
telencephalon where the concentration of Pax6 reaches sufficient levels, i.e.
a medioventral domain of the spinal cord, and a lateral domain of the cerebral
cortex (Fig. 1). E1 can be
ectopically activated in regions where Pax6 concentration is normally low but
has been artificially raised either by electroporation of a Pax6 expression
construct (Fig. 7), or by
introduction of multiple copies of a yeast artificial chromosome containing
the Pax6 gene (Fig.
6).
Pax6 has therefore an essential role in determining the size of the domain
of activity of one of the enhancers of Ngn2, and it may thus be
involved in establishing borders of Ngn2 expression, in particular at
the sulcus limitans in the spinal cord and at the striatal-cortical border in
the telencephalon. Although it is well established that Pax6 is an important
regulator of neural cell fates (Ericson et
al., 1997; Takahashi and
Osumi, 2002
), the significance of its non-uniform, graded
expression along the dorsoventral axis of the spinal cord, has remained
unclear. We present evidence that this gradient is involved in controlling the
spatial pattern of expression of one of its targets, Ngn2.
The concentration gradient of Pax6 in the neocortex, from high
rostrolateral to low caudomedial, has been shown to be important for its
regionalisation in distinct areas, as shown by the analysis of Pax6 mutant
embryos in which rostral cortical areas contract while caudal areas expend
(Bishop et al., 2000). The HD
protein Emx2 and the nuclear receptor COUP-TFI, are also distributed in
gradients across the neocortex, and mutant analysis has similarly implicated
these factors in regionalisation of this territory
(Bishop et al., 2000
;
Mallamaci et al., 2000
;
Zhou et al., 2001b
). How
concentration gradients of transcription factors translate into discrete
cortical areas having unique molecular, architectonic and functional
properties is currently not known. Our results on the regulation of the E1
enhancer suggest that factors such as Pax6 and Emx2 could directly activate
the expression of target genes involved in specification of area identity in
restricted domains of the neocortex.
The role of the low affinity Pax6 binding site in establishing the
domain of E1 activity
Our results support a model whereby the ability of the E1 element to only
respond to high concentrations of Pax6 protein is due to the presence of a low
affinity binding site occupied only when the concentration of Pax6 reaches a
high level. The low affinity of the E1.1 sequence was demonstrated by the
following observations. Compared with a consensus Pax6 binding sequence, the
E1.1 sequence only forms a small amount of complex with recombinant Pax6
protein in vitro (Fig. 3).
Moreover, 4 tandem copies of E1.1 cannot recruit enough Pax6 protein to
efficiently activate a basal promoter in a context where Pax6 is expressed at
low levels as in the early neural tube, whereas the same construct is
activated by high levels of exogenous Pax6 protein
(Fig. 3). Evidence that the low
affinity of the E1.1 site underlies the property of the E1 element to respond
solely to high Pax6 levels, is that increasing the affinity of this site
results in an expansion of the E1 domain into regions of low Pax6 expression
(Figs 6,
7).
In invertebrate species several examples are known of transcription factors
activating only a subset of their target genes at a particular concentration.
For example, the transcription factor PHA-4 has been shown to sequentially
activate a number of pharyngeal genes in C. elegans, through the
progressive increase in PHA-4 concentration during development, and the
presence in target genes of binding sites with different affinities for PHA-4
(Gaudet and Mango, 2002).
Thus, the affinity of binding sites determines a temporal pattern of gene
expression in this case, and a spatial expression pattern in the case of the
interaction between Pax6 and E1. Other mechanisms, such as cooperative DNA
binding, have been implicated in the establishment of gene expression patterns
by gradients of transcription factors. Further study of the regulation of
Ngn2 should determine whether diverse strategies are similarly used
to establish the complex expression patterns of proneural genes.
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ACKNOWLEDGMENTS |
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