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First published online 4 April 2007
doi: 10.1242/dev.02845
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Center for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, 20 Marie Curie, K1N 6N5, Ottawa, Ontario, Canada.
* Author for correspondence (e-mail: Mekker{at}uottawa.ca)
Accepted 26 February 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Telencephalon, Diencephalon, Interneuron, Homeobox, Enhancer, Distal-less, bHLH, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Recent studies have revealed that the developing vertebrate brain can be
subdivided into multiple regions based on genetic interactions between genes
involved in neural differentiation (Manuel
and Price, 2005; Pasini and
Wilkinson, 2002; Trujillo et
al., 2005). The fate of neuronal progenitors present in these
defined regions is controlled by distinct regional molecular microenvironments
that give them their own identity. Thus, characterization of the different
genetic interactions controlling the differentiation of the neuronal
progenitors will provide new insights in the understanding of brain
development and various neurological disorders.
The homeobox genes of the Dlx family, which encode homeodomain-containing
transcription factors, play an important role during the development of the
forebrain (Bendall and Abate-Shen,
2000; Panganiban and
Rubenstein, 2002; Zerucha and
Ekker, 2000). This family comprises six members in mammals
(Stock et al., 1996),
organized as three bigene clusters - Dlx1/2, Dlx3/4 and
Dlx5/6 - in an inverted and convergent configuration. Of the six
mammalian Dlx genes, only Dlx1, Dlx2, Dlx5 and Dlx6 are
expressed in the telencephalon and in the diencephalon
(Bulfone et al., 1993;
Robinson et al., 1991;
Liu et al., 1997;
Eisenstat et al., 1999).
Expression of the Dlx genes in the telencephalon is restricted to the
differentiating GABAergic (
-aminobutyric acid releasing) projection
neurons and interneurons (Anderson et al.,
1997b; Stuehmer et al.,
2002a; Stuehmer et al.,
2002b). Most of these neurons are born in the ventricular and
subventricular zones (VZ and SVZ, respectively) of the lateral and medial
ganglionic eminences (LGE and MGE, respectively). Soon after, they undergo a
radial or a tangential migration through the SVZ to their final destinations
in the piriform, cerebral cortex, hippocampus and olfactory bulb
(Anderson et al., 1997a;
Chapouton et al., 1999;
de Carlos et al., 1996;
Lavdas et al., 1999;
Sussel et al., 1999;
Tamamaki et al., 1997;
Tamamaki et al., 1999;
Wichterle et al., 1999). Dlx
genes have highly overlapping but distinct patterns of forebrain expression.
As reviewed by Panganiban and Rubenstein, Dlx2 is mainly expressed in
the VZ and SVZ of the MGE and LGE of the telencephalon of E12.5 mouse embryos,
where early differentiation occurs
(Panganiban and Rubenstein,
2002). Dlx1 expression is found in all three zones
(ventricular, subventricular and mantle). Expression of Dlx5 and
Dlx6 is confined to the more-differentiated migrating neurons found
in the subventricular and mantle zones.
The inactivation of individual Dlx genes implicated in forebrain
development results in a subtle phenotype
(Acampora et al., 1999;
Anderson et al., 1997b;
Qiu et al., 1997;
Qiu et al., 1995;
Robledo et al., 2002). For
example, mice lacking Dlx1 display a reduction in
calretinin+ (also known as calbindin 2 - Mouse Genome Informatics)
and somatostatin+ interneuron subtypes
(Cobos et al., 2005). Mutant
mice harboring this subtype-specific loss of interneurons show behavioral and
histological signs of epilepsy. Inactivating the function of both
Dlx1 and Dlx2 results in a massive reduction of GABAergic
interneurons of the cerebral cortex, which is mainly due to a lack of
tangential migration of the immature interneurons born in the VZ and SVZ of
the ventral telencephalon (Anderson et al.,
1997a). These mice also show a reduction in the number of
interneurons in the striatal and in the olfactory bulb.
Interestingly, in Dlx1 Dlx2 double mutants, the expression of the
Dlx5 and Dlx6 genes is reduced. The DLX2 protein has been
shown to bind to the I56i sequence, an enhancer found in the intergenic region
of the Dlx5/6 bigene cluster
(Zerucha et al., 2000). This
finding was supported by chromatin immunoprecipitation studies
(Zhou et al., 2004).
Phylogenetic footprinting and transgenic analyses have also revealed the
existence of at least two cis-acting regulatory elements, called I12a and
I12b, in the intergenic region separating Dlx1 and Dlx2
(Ghanem et al., 2003;
Park et al., 2004). We also
found one conserved cis-acting regulatory element, URE2, in the 5'
flanking region of Dlx1 (Hamilton
et al., 2005).
Of the cis-acting regulatory elements identified thus far in the
Dlx1/2 and Dlx5/6 bigene clusters, four - URE2, I12b, I56i
and I56ii - contribute to Dlx gene expression in the forebrain. Analysis of
reporter gene constructs in transgenic mice indicated that the activity of the
I56i, I12b and URE2 enhancers was very similar when examined on whole-mount
preparations (Ghanem et al.,
2003) (N.G. and M.E., unpublished). However, in-depth analysis of
sections from these transgenic embryos at different stages of development
revealed subtle differences in the expression of the transgenes driven by each
of the cis-acting elements (N.G., M. Yu, J. Long, G.H., J. L. R. Rubenstein
and M.E., unpublished).
Despite their overlapping activities, the various forebrain enhancers are
highly divergent in their sequence (Ghanem
et al., 2003; Zerucha et al.,
2000), suggesting that they could respond to distinct trans-acting
factors. In an attempt to characterize the genetic pathways responsible for
the expression of the Dlx gene family in the forebrain, we performed DNase I
footprinting analysis of I12b. Mutations were introduced into the I12b
enhancer at each putative forebrain transcription factor-binding site
identified by DNase I footprinting and these mutant enhancers were used in a
transgenic assay. Our results suggest that Dlx1/2 expression is auto-
or cross-regulated by DLX proteins in the telencephalon and diencephalon. We
also provide evidence for the regulation of Dlx1/2 expression by
transcription factors such as MASH1 (also known as ASCL1 - Mouse Genome
Informatics), MEIS1 and MEIS2 (also known as MRG1 - Mouse Genome
Informatics).
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
In addition, the protein extract was dialyzed against the resuspension buffer
using a Spectra/Por dialysis cuvette (VWR) overnight at 4°C. Aliquots of
50 µL were frozen in liquid nitrogen and stored at -80°C.
DNase I footprinting analysis
To obtain an optimal footprinting resolution, the I12b enhancer was divided
into two overlapping fragments (nucleotides 1-292 and 259-437; see
Fig. 2B). The primers used to
amplify the two fragments were: mI12b-1
(5'-GGAATTCGCGTACAGCTGCAAACC-3') and mI12b-292
(5'-CGGTACCTACCGGAGAATTGCAGAG-3'); mI12b-259
(5'-GGAATTCAATAGGTGCGAGCTGCCA-3') and mI12b-437
(5'-CGGTACCAGTGAGGGAAAGGTTGGG-3'); restriction sites
(underlined) were incorporated at the 5' (EcoRI) and 3'
(KpnI) ends of each fragment. PCR fragments were cloned into the
pDrive vector (Qiagen). Each fragment was recovered by digestion with
EcoRI and KpnI followed by gel purification (QIAquick Gel
Extraction Kit, Qiagen). Directional labeling of each fragment was performed
by 5' end-fill using the large fragment of DNA polymerase I
(Invitrogen). Labeled fragments were then purified from low melting point
agarose gels using ß-agarase (New England Biolabs).
Binding reactions (50 µL) comprised 25 µL of binding buffer [20%
glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, 20 mM HEPES pH 7.9, 4% poly(vinyl
alcohol)], 0.5 µL of 1 mg/ml poly(dAdT) (Sigma), 2-3 ng of end-labeled DNA
fragment, nuclear extract and 60 mM KCl. The reaction mixtures were incubated
on ice for 20 minutes. One volume of 10 mM MgCl2 5 mM
CaCl2 was added. Each reaction mixture was then treated with
increasing amounts of DNase I, from 0.01 to 0.1 Kunitz units (Worthington) for
120 seconds and stopped by adding 100 µL STOP solution (1% sodium dodecyl
sulphate, 200 mM NaCl, 20 mM EDTA pH 8.0, 40 mg/mL tRNA). Reactions were
extracted twice with one volume of phenol-chloroform, once with one volume of
chloroform and precipitated with two volumes of ethanol at -80°C for 20
minutes. Reactions were centrifuged for 15 minutes at 10,000
g. DNA pellets were washed with 80% ethanol and dried in a
vacuum desiccator. Pellets were resuspended in 10 µL of loading buffer (95%
formamide, 20 mM EDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol FF).
Reactions were loaded on a 6% (19:1 acrylamide:bis-acrylamide) polyacrylamide
sequencing gel containing 7 M urea and 1xTBE (89 mM Tris base, 89 mM
boric acid, 2.5 mM EDTA). The gel was run at 80 volts in 1xTBE and
dried. Radioactive bands were visualized by phosphorimaging (Molecular
Dynamics). Guanine+adenine chemical sequencing was performed as previously
described (Maxam and Gilbert,
1980).
The sequences of protected regions were compared with a library of matrix descriptions for transcription factor-binding sites using MatInspector software (Genomatix).
Co-transfection experiments
A zebrafish dlx2a cDNA (845 bp) encompassing the full-length
coding sequence was PCR-amplified and cloned into the EcoRI site of
the pTL2 or pcDNA3/HisB expression vector
(Zerucha et al., 2000). A
Dlx2a homeodomain mutant was obtained by changing essential amino acids of the
third loop of the homeodomain: Trp170, Phe171, Gln172, Asn173, Arg174 and
Arg175 were changed to glycine using overlapping PCR.
The mouse I12b enhancer was PCR-amplified and inserted upstream of the
thymidine kinase (tk) minimal promoter driving expression of
the chloramphenicol acetyltransferase (CAT) gene (pBLCAT2 vector)
(Luckow and Schutz, 1987).
zI56i-pBLCAT2 (Zerucha et al.,
2000) was used as positive control. MASH1 and E47-pCDNA3
constructs were kindly provided by François Guillemot (National
Institute for Medical Research, London, UK). Cell culture, transfection and
the CAT assay were carried out as previously described
(Zerucha et al., 2000).
Experiments were performed in duplicate and repeated a minimum of three
times.
Mutagenesis
Mutant enhancers were generated by PCR using overlapping fragments. A mix
(1:3) of Taq and Pfx DNA polymerases (Invitrogen) was used to avoid unwanted
mutations. A first fragment was amplified from the I12b enhancer
(I12b-pBluescript®KS) using oligonucleotides I12b-437
(5'-CGGTACCAGTGAGGGAAAGGTTGGG-3') and T7 primer (annealing site in
pBluescript vector). A second overlapping fragment was generated with an
oligonucleotide containing the second mutation and an adaptor sequence
corresponding to the SP6 promoter (mutations are underlined): Mut FP1,
5'-CTGAAATTACTGTCTAGATGTTGTCTTTGA-3'; Mut FP2,
5'-AACTGCATTAGAGGAGCTCAACCTGAAATTA-3';
Mut FP3, 5'-ATGGGGAAACTGCTCTAGAATAAATAAACCTG-3'; Mut
FP4, 5'-GCGAAAAAATTGCTCATCTAGACCAGAGAGATGGG-3'; Mut
FP5,
5'-ATACTCTAGAATGGAGCTCGCGGGCTCCGGTA-3';
and Mut FP6,
5'-GCTAAGTCTGTCTTCTAGACTTCGCTGCTGTGC-3'.
After purification from agarose gels, the two fragments were used as template for a final PCR with SP6 and T7 primers. Finally, the mutant enhancer fragments were purified from agarose gels, TA-cloned and sequenced.
Transgenic experiments
The I12b mutant enhancers were subcloned into p1230 vector
(Yee and Rigby, 1993) that
contains a human ß-globin minimal promoter and lacZ reporter
cassette. Transgenic animals were produced and analyzed as previously
described (Zerucha et al.,
2000). For each construct, at least three primary transgenic
embryos were obtained. Embryos shown in Figs
4,
5 and
6 are representative transgenic
animals of two or more independent integration events.
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The complementary oligonucleotides were annealed and end-labeled using
[32P]-ATP (Amersham) and T4 polynucleotide kinase (New
England Biolabs). Electromobility shift assay (EMSA) reactions were performed
by incubating 1 ng of labeled DNA with recombinant MASH1 and E47 proteins (TNT
Quick Coupled Transcription/Translation Kit, Promega) or GST-Dlx2a in reaction
buffer (7 mM Tris pH 7.5, 81 mM NaCl, 2.75 mM dithiothreitol, 5 mM
MgCl2, 0.05% NP-40, 1 mg/mL bovine serum albumin, 25 µg/mL
poly(dIdC), 10% glycerol) at 0°C for 30 minutes. Reactions were loaded on
a polyacrylamide gel (6% acrylamide, 0.16% bis-acrylamide) in 0.5xTBE.
The gel was run at 80 volts in 0.5xTBE and dried. Radioactive bands were
visualized by phosphorimaging (Molecular Dynamics).
For supershift assays, nuclear extracts were preincubated overnight at 4°C with increasing amounts (100 ng, 1 µg, 3 µg) of anti-MASH1 and anti-MEIS1 antibodies (sc-13222X and sc-25412X, Santa Cruz Biotechnology).
Chromatin immunoprecipitation
The LGE and MGE were dissected from E11.5 mouse telencephalon. Chromatin
extraction and immunoprecipitation were performed essentially as previously
described (Mac et al., 2000)
with a few modifications. First, tissues were disaggregated using a dounce
homogenizer and were resuspended in cell lysis buffer (50 mM Hepes pH 8, 75 mM
KCl, 0.5% NP-40, plus protease inhibitors) prior to nuclei release. Second,
protein-A sepharose (Sigma, P3391) was used for the immunoprecipitation.
Third, chromatin was precipitated using 1 µg of anti-MASH1 antibody (Santa
Cruz). Washing and elution were carried out as previously described
(Boyd and Farnham, 1997). After
elution, reactions were incubated at 65°C for 4 hours to reverse the
DNA-protein cross-link, then treated with proteinase K (Invitrogen), extracted
with phenol-chloroform and precipitated with ethanol. DNA was collected by
centrifugation and resuspended in 100 µL of H2O and analyzed by
PCR. The I12b and Hes6 enhancers were amplified using the following primers:
mI12b-ChIP.For (5'-GAGGGTCAGCATCATTTCAC-3') and mI12b-ChIP.Rev
(5'-GCAAGCTGTGGACCATAG-3'); mHes6-ChIP.For
(5'-GCCGAGGCTGCTGTCTG-3') and mHes6-ChIP.Rev
(5'-TGGGCGTCTGGCCGACA-3'). After 28-30 cycles, PCR products were
analyzed on ethidium bromide-stained agarose gels.
Sequence analysis
The mouse and human Hes6 sequences were obtained from public
databases
(http://www.ensembl.org):
mouse Hes6, Ensembl gene ID ENSMUSG00000067071; human HES6,
Ensembl gene ID ENSG00000144485. Pairwise sequence alignment was performed
with PipMaker (available at
http://bio.cse.psu.edu/pipmaker/).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Zerucha et al.,
2000). Initial characterization of the I12b enhancer activity in
transgenic mice indicated that this enhancer targets reporter gene expression
exclusively to the forebrain (Ghanem et
al., 2003) (Fig.
1B). Enhancer activity of I12b is first detectable at E10 in the
diencephalon, followed by expression in the basal telencephalon at E10.5 (data
not shown). Transverse sections of telencephalon at E11.5 revealed
lacZ expression in the subventricular and mantle zones of the LGE and
MGE, but also in the anterior entopeduncular area (AEP), the preoptic area and
the suprachiasmatic band (SCB). (Fig.
1C) (N.G. and M.E., unpublished). The I12b-lacZ reporter
transgene persisted in the SVZ of the LGE and MGE from E12.5 to E15.5 and
reporter gene expression was found in cells migrating to the dorsal pallium
(Fig. 1D). Expression of
I12b-lacZ was still detected in the neocortex after birth (P0) and at
P25 (in the somatosensory cortex), the latest stage tested
(Fig. 1E-G). However,
expression of the transgene in the septum and the striatum was much reduced at
this stage (Fig. 1E-G, Sp and
St). At P25, expression of the transgene is also observed in all layers of the
olfactory bulb containing GABAergic neurons (data not shown). To better define the important sequence elements within I12b and as a first step in the identification of trans-acting factors binding to this enhancer, we performed DNase I footprinting experiments using nuclear extracts from E13.5 mouse embryonic forebrain. Two overlapping DNA fragments from the I12b enhancer (Fig. 2B) were used. DNase I footprinting revealed six protein-DNA interactions (Fig. 2A, FP1 to FP6). The sequence of these protected regions (Fig. 2B) were compared with a library of matrix descriptions for transcription factor-binding sites using MatInspector software. The position of the protected area on the enhancer sequence and relevance to telencephalic development were used as the main criteria to discriminate between the 87 candidate transcription factor-binding sites found by MatInspector.
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;
Zhou et al., 2004). To test
the hypothesis that DLX2 can bind and activate transcription through the FP1
and FP4 binding sites present in I12b, a transfection reporter assay was
performed. P19 murine embryonic carcinoma cells were co-transfected with a
reporter construct in which the CAT reporter gene was placed under the control
of the mouse I12b enhancer (mI12b-pBLCAT2). A plasmid expressing the zebrafish
Dlx2a protein (Dlx2-pTL2) was co-transfected. In the presence of Dlx2a, an
11-fold increase in relative CAT activity was observed
(Fig. 3A). Similar results were
also obtained when vectors expressing other zebrafish Dlx proteins (Dlx1a,
Dlx3b, Dlx5a, Dlx6a) were used in this assay (data not shown). This activation
is dependent on the DLX homeodomain because transfection of a Dlx2a
homeodomain mutant (W170G, F171G, Q172G, N173G, R174G and R175G) did not show
any transcriptional activation in the reporter assay
(Fig. 3B). Furthermore,
recombinant Dlx2a synthesized in vitro was shown to bind to a double-stranded
oligonucleotide corresponding to FP4 in an EMSA (see Fig. S1 in the
supplementary material). Taken together, our data show that DLX proteins are
able to bind to and activate transcription from the I12b enhancer.
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).
Analysis of the sequences surrounding FP5 suggests the presence of an E-box
element - a sequence recognized by a subset of basic helix-loop-helix (bHLH)
proteins including the products of the neurogenin (Neurog), NeuroD, Mash
(Ascl), Olig and E (Tcfe) gene families (reviewed by
Ross et al., 2003). Three of
these bHLH factors have been shown to be expressed in the telencephalon:
neurogenin 1 (Neurog1), neurogenin 2 (Neurog2) and the
mammalian achaete-scute homolog 1, Mash1 (Ascl1)
(Guillemot and Joyner, 1993;
Lo et al., 1991;
Sommer et al., 1996).
Mash1 controls the transition of the multipotent cortical progenitors
from proliferation to neurogenesis (Cau et
al., 1997; Guillemot et al.,
1993; Hirsch et al.,
1998; Sommer et al.,
1995). Mash1 is expressed in the proliferative zone of
the LGE and MGE of the telencephalon. Many studies provide evidence suggesting
that this proneural factor is a potential upstream regulator of Dlx2,
but so far only genetic evidence supports this hypothesis
(Casarosa et al., 1999;
Fode et al., 2000;
Horton et al., 1999;
Letinic et al., 2002;
Porteus et al., 1994;
Yun et al., 2002). First,
Mash1 and Dlx1/2 show overlapping patterns of expression in
the VZ and SVZ of the ventral telencephalon
(Porteus et al., 1994).
Second, Horton and collaborators have shown that mice lacking Mash1
have a reduced number of cells expressing Dlx genes in the SVZ of the
ganglionic eminence at E12.5 (Horton et
al., 1999). Third, ectopic expression of Mash1 leads to
an upregulation of Dlx1/2 in the neocortical neurons
(Fode et al., 2000). However,
evidence of a direct regulation of Dlx1/2 by the MASH1 protein has
yet to be demonstrated at the molecular level.
The MatInspector software identified FP6 as possible binding site for yet another homeodomain-containing transcription factor. Sequence comparisons between different species show that the FP6 homeobox (TAAT) is present in human and mouse, but not in the zebrafish I12b sequence (Fig. 2B).
Mutational analysis of putative binding sites in I12b
To investigate the functionality of the putative transcription
factor-binding sites found in the I12b enhancer, and to understand their
relative contributions to Dlx gene regulation in the forebrain, we tested the
effect of mutating these sites in a transgene reporter assay. Mutations
affecting 2-8 nucleotides were introduced for each putative binding site
(Fig. 2C). Mutated enhancers
were subcloned upstream of a ß-globin minimal promoter-lacZ
cassette (Yee and Rigby,
1993). These constructs were injected into fertilized mouse eggs
to produce transgenic animals. Staining of E11.5 primary transgenic mouse
embryos revealed that mutagenesis of each of the DLX/MSX/NKX2.5 (FP1 and FP4)
or MASH1 (FP5) binding sites resulted in a reduction of the lacZ
expression in the ventral telencephalon
(Fig. 4, compare A,B,E,F).
Transverse sections through the brain of the transgenic embryos indicated that
the mutations impair enhancer activity in both the LGE and MGE of the ventral
telencephalon (Fig. 5, compare
C,D,I-L with A,B). The effects of the mutations appeared to be stronger in the
more-rostral cells (Figs 4,
5).
In embryos carrying the 
FP1-I12b-lacZ reporter transgene
(affecting one of the two DLX/MSX/NKX2.5 sites), expression was observed in
only a subset of cells located at the border between the ventral MGE and AEP
(Fig. 5D). Expression was also
observed in the SCB in the diencephalon
(Fig. 5D, arrow). Reporter gene
expression in primary transgenic embryos carrying the
FP4-I12b-lacZ transgene (affecting the second DLX-binding
site, Fig. 5J) or the
FP5-I12b-lacZ transgene (affecting the MASH1-binding site,
Fig. 5L) was scattered
throughout the superficial mantle zone of the MGE and intermediate zone and in
the AEP. All three mutations abolished ß-galactosidase activity in the
rostral LGE (Fig. 4A,B,E,F and
Fig. 5A,C,I,K).
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FP1-I12b-lacZ
or FP4-I12b-lacZ transgenes showed an overall reduction in
reporter gene expression in the LGE and MGE and reduced expression in
interneurons migrating to the cortex (data not shown). Interestingly, E13.5
transgenic embryos carrying FP2 (MEIS1/2), FP5
(MASH1) or FP6 (HD TF)-I12b-lacZ transgenes showed
full restoration of the expression obtained with the wild-type mI12b enhancer
construct (I12b-lacZ), suggesting that these sites play a role in
establishing Dlx1/2 expression, whereas DLX-binding sites (FP1 and
FP4) are responsible for maintaining this expression (data not shown).
In the diencephalon, lacZ expression driven by
FP1-I12b-lacZ (Fig.
6B) was reduced compared with that driven by the wild-type I12b
sequence (Fig. 6A). This
reduction occurred in both domains of expression, the prethalamus and the SCB.
In the FP4-I12b-lacZ and FP5-I12b-lacZ
transgenic embryos, lacZ expression was absent in the prethalamus and
reduced in the SCB as compared with the wild-type enhancer
(Fig. 6E,F, arrows).
Transgenic embryos produced with the FP2-I12b-lacZ
construct displayed a slight reduction in lacZ expression in the
ventral telencephalon as compared with wild-type I12b-lacZ, and an
almost complete abolition of reporter expression in the diencephalon
(Fig. 4C). Transverse sections
of the ventral telencephalon revealed that lacZ expression was weaker
in the MGE and AEP and was absent in the LGE and preoptic area
(Fig. 5E,F). Expression in the
MGE was concentrated at the intermediate zone. An overall reduction in
lacZ staining was found in the diencephalon
(Fig. 6C). Reduction in
expression mainly occurred in the prethalamus
(Fig. 6C, arrow). However, the
effect of FP2 mutation on the targeted activity of the I12b enhancer to the
diencephalon was variable, as two independently generated
FP2-I12b-lacZ transgenic embryos showed different levels of
reduction (Fig. 4C,
Fig. 6C).
Mutagenesis of FP3 resulted in a relative increase in reporter transgene
expression in all domains compared with the wild-type I12b-lacZ
transgene (Fig. 4D,
Fig. 5G,H,
Fig. 6D). For example, the
faint frontonasal prominence staining present in the control embryo
(Fig. 4A) was greatly increased
following mutagenesis of the FP3 site (Fig.
4D, arrow). This increase in staining was not the result of an
integration effect, as three FP3-I12b-lacZ transgenic embryos
obtained from two separate experiments gave similar staining. This result
suggests that the FP3 site is probably recognized by a homeodomain-containing
repressor.
Primary transgenic embryos carrying the FP6-I12b-lacZ
construct showed a reduction in lacZ staining in particular cells
(Fig. 4G,
Fig. 5M,N,
Fig. 6G). This reduction gave a
granular aspect to the staining as compared with the wild-type
I12b-lacZ embryos (Fig.
5B,N). Since Dlx1 and Dlx2 are expressed in at
least four different subtypes of interneurons, the granular staining could be
the result of a subtype-specific loss in which a mutation in FP6 affects one
or more subtypes of interneurons. The same type of granular staining was
observed in the diencephalon (Fig.
6G). Although this granular expression could correspond to
specific progenitor populations, the paucity of specific markers at this early
stage makes it difficult to confirm such a correlation.
A functional bHLH-binding site is present in the I12b enhancer
We found an E-box sequence in the I12b enhancer (FP5). To test the
hypothesis that FP5 is a functional E-box and to determine if MASH1 is able to
bind to that sequence and activate transcription through this site, we
performed co-transfection and EMSAs. A plasmid expressing Mash1 was
transfected with a mouse I12b enhancer reporter construct (mI12b-pBLCAT2) into
P19 murine embryonic carcinoma cells. In the presence of MASH1, a 4-fold
increase in relative CAT activity was observed
(Fig. 7A).
We performed an EMSA using a 27 bp double-stranded oligonucleotide corresponding to the FP5 sequence, which was incubated with recombinant MASH1 protein and its co-factor E47 (also known as TCFE2A - Mouse Genome Informatics) produced by in vitro transcription/translation. Up to three protein-DNA complexes were observed on the EMSA (Fig. 7B, lanes 2-4, arrowheads). To further prove that the E-box present in I12b is a functional binding site, we used an oligonucleotide that lacked the site as a result of introducing the same mutation that was used in the transgenic experiments shown in Figs 4, 5 and 6. Lower motility complexes were not obtained with the mutant fragment in the presence of MASH1 and E47 in vitro translation extracts (Fig. 7B, compare lanes 6-8 with lanes 2-4). In a supershift assay, MASH1-E47-FP5 complex was completely eliminated by preincubation with an anti-MASH1 antibody, whereas in the control experiment using a non-specific antibody these complexes remained intact (Fig. 7C, compare lanes 7-9 with lanes 3-5, arrowheads).
|
;
Murre et al., 1989). In our
experiments, the recombinant MASH1 bound to FP5 with almost equal efficiency
in the absence or presence of E47 (data not shown). Thus, MASH1 is likely to
form a heterodimer preferentially with a co-factor present in the in vitro
translation extract whose identity remains to be determined.
To determine if MASH1 is able to bind to the I12b enhancer in vivo, we
developed a chromatin immunoprecipitation assay (ChIP) for this protein. To
our knowledge, this procedure has not previously been used for MASH1 and
little is known about MASH1-binding sites in vivo. Therefore, we also tested
our ChIP assay on an independent site. We used a conserved sequence that has
been suggested as the site of Hes6 gene regulation by MASH1 in mouse
prostate cells (Hu et al.,
2004). Interestingly, Hes6 is also expressed in the
developing telencephalon and binding of MASH1 to Hes6 regulatory
element(s) should be detectable in the chromatin we isolated in our ChIP
analysis of MASH1 with I12b. Phylogenetic footprinting analysis of the
Hes6 promoter region allowed us to identify a 217 bp conserved
element (Hes6 enh) located 671 bp from the mouse Hes6 exon1 (see Fig.
S2A in the supplementary material). Three conserved E-box sequences were found
in this element (see Fig. S2B in the supplementary material). In a ChIP assay
performed on fixed chromatin isolated from the LGE and MGE of E11.5 mouse
embryos, both the I12b enhancer and the conserved Hes6 enh sequence were
precipitated with the anti-MASH1 antibody
(Fig. 8). Thus, MASH1 is able
to bind, in vitro and in vivo, to the I12b enhancer.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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DLX proteins as factors necessary for optimal I12b enhancer activity
Two sequences in I12b, FP1 and FP4, are potential binding sites for DLX and
MSX proteins. They could also correspond to a low affinity NKX2.5-binding site
(5'-ATAATTA-3'). FP1 and FP4 completely fulfill the DLX/MSX
consensus binding site requirement, G-A/C-TAATT-A/G-G/C
(Feledy et al., 1999).
Co-transfection assays provided evidence that DLX proteins activate
transcription from the I12b enhancer and that this is dependent on the
presence of the homeodomain (Fig.
3). DLXIN-1 (MAGED1 - Mouse Genome Informatics), GRIP and MSX
proteins (Masuda et al., 2001;
Yu et al., 2001;
Zhang et al., 1997) have been
proposed as interacting partners for DLX proteins. For example, it has been
shown that DLX and MSX proteins could form homo- and heterodimers
(Zhang et al., 1997). Msx
genes are expressed in the most dorsal parts of the forebrain where the Dlx
genes are not expressed, suggesting that these transcription factors are
unlikely to play a role in Dlx gene regulation. We cannot exclude
co-expression of Msx and Dlx genes at the neural plate stage in the neural
ridge.
|
|
).
Transcripts of the Nkx2.5 gene are found in early cardiac cell
progenitors, but not in the developing brain
(Komuro and Izumo, 1993).
However, another member of the Nkx2 family, Nkx2.1, was shown to play
a role in the development of the ventral telencephalon. Nkx2.1 (also
known as thyroid transcription factor 1, Titf1), is expressed in the
developing thyroid, telencephalon, and lung
(Guazzi et al., 1990;
Lazzaro et al., 1991;
Price et al., 1992). In the
developing telencephalon, Nkx2.1 expression is restricted to the MGE
of the basal telencephalon as well as to parts of the septum, the
entopeduncular area and preoptic area
(Sussel et al., 1999). NKX2.1
has been shown to have high affinity for the 5'-CAAG-3' core motif
and to inefficiently bind a general homeodomain core sequence
(5'-TAAT-3') resembling FP1 and FP4
(Damante et al., 1994).
Extrapolating from these data, we can propose that NKX2.1 could bind, with low
affinity, to the FP1 and FP4 binding sites of the I12b enhancer, but this
assertion remains to be confirmed.
The FP1 and FP4 DLX/MSX/NKX2.5-binding sites in the I12b enhancer seem to
play a crucial role in Dlx gene regulation. Several areas of I12b reporter
transgene expression disappear when individual binding sites are mutated.
Reductions in expression were also observed at E13.5. When mutations were
introduced in both DLX/MSX/NKX2.5-binding sites, the reporter transgene
expression targeted by I12b was totally abolished (data not shown).
Interestingly, E13.5 transgenic embryos carrying I12b reporter transgenes with
mutagenized MEIS1/2 or MASH1 sites showed transgene expression comparable to
that obtained with the wild-type enhancer. Thus, we propose that
Dlx1/2 expression in the ventral telencephalon is first activated by
neural determinants involving some of the sites we identified. Then,
expression of Dlx1Dlx/2 would be maintained by the newly synthesized
DLX1 and/or DLX2 proteins through a positive-feedback loop. Products of the
Dlx5 and Dlx6 paralogous genes, whose expression follows
that of Dlx1/2 chronologically
(Liu et al., 1997) and depends
on Dlx1/2 function (Anderson et
al., 1997b; Zerucha et al.,
2000), could also act as positive regulators.
|
;
Zhou et al., 2004). The
vertebrate Dlx bigene clusters are thought to have originated from
duplications of an ancestral cluster
(Stock et al., 1996;
Zerucha and Ekker, 2000).
Thus, cis-regulatory elements in the Dlx1/2 and Dlx5/6 loci
might descend from a cis-acting element that existed in the ancestral Dlx
bigene cluster. Nucleotide sequence comparisons between I12b and I56i enhancer
sequences revealed that two DLX/MSX/NKX2.5 sites are present in both enhancers
and the physical distance separating the two sites is similar for both
enhancers (Ghanem et al.,
2003). Footprinting analysis of the I56i enhancer revealed
protein-DNA interactions at the two binding sites (L.P., N. Shipley and M.E.,
unpublished). However, the rest of the I12b and I56i enhancer sequences (more
than 400 bp) are very different. Therefore, although it seems as if I12b and
I56i may function through partially similar mechanisms involving auto- or
cross-regulation by DLX proteins, it is not possible to say at this time
whether the two enhancers result from the duplication of an ancestral
regulatory element.
The potential role of the MEIS2 in Dlx1/2 gene regulation
Meis transcription factors are potent regulators of cell proliferation. Of
the three mammalian Meis genes, only Meis1 and Meis2 have
been shown to be expressed during telencephalic development
(Cecconi et al., 1997;
Oulad-Abdelghani et al., 1997;
Toresson et al., 1999).
Meis1 transcripts are mainly present at the lateral edges of the LGE
and MGE in E12.5 mouse embryos, whereas Meis2 was found to be highly
expressed in the SVZ of the LGE at the same stage. In the LGE, Meis2
expression is restricted to the proliferating progenitors
(Toresson et al., 1999). In
the diencephalon of E11.5 embryos, Meis1 is expressed in the optic
stalk and the prosomere 1, whereas Meis2 transcripts are present in
the ventral thalamus. We have shown that a potential MEIS-binding sequence is
present in I12b. The MatInspector software identified this site as a
MEIS1-binding site. However, it was recently reported that MEIS2 is able to
recognize the same sequence (Yang et al.,
2000). Our transgenic experiments provided evidence that the
putative MEIS site sequence contributes to I12b enhancer activity, more
specifically in the LGE of the ventral telencephalon. Based on the expression
pattern of the Meis genes, we can assume that MEIS2, but not MEIS1, is likely
to be involved in this regulation. Toresson and collaborators showed that
MEIS, PBX and DLX proteins colocalize in the SVZ of the LGE but not the MGE at
E12.5 (Toresson et al., 2000).
The FP3 site is a homeobox binding site, located not far from the MEIS FP2
site. We hypothesize that FP3 could be a binding site for a MEIS1 co-factor.
However, sequence comparison of the PBX consensus binding site with the FP3
sequence did not reveal any similarities.
A bHLH factor binds to an E-box in the intergenic I12b enhancer
Neurogenesis in the ventral telencephalon is a balancing act controlled in
part by repressor-type and activator-type bHLH transcription factors. Neural
stem cells are kept in an undifferentiated state by repressor-type bHLH
proteins (for example, members of the HES family). In the absence of the
repressor-type bHLHs, expression of the activator-type bHLHs is upregulated,
leading to the commitment of multipotent stem cells to a neuronal cell
lineage. Expression of the activator-type Mash1 promotes neurogenesis
of progenitors present in the VZ and SVZ of the ventral telencephalon. These
committed neural progenitors will then be specified into multiple neuronal
lineages, including various subtypes of GABA-expressing interneurons and
projection neurons. Mash1 has a dual function in cell fate
specification and differentiation of ventral telencephalon progenitors
(Yun et al., 2002). First,
through a non-cell-autonomous function, Mash1 delays the maturation
of neighboring progenitors by using the Notch signaling pathway.
Differentiation of these delayed progenitor cells gives rise to a population
of cells called late-born neurons. Mash1 also has a cell-autonomous
function in the subcortical telencephalon. Mash1 expression triggers
the differentiation process of some progenitor cells. Dlx1/2 are
early markers of early-born neurons and their expression quickly follows that
of Mash1. As proposed by previous genetic studies, Mash1
could be a potential regulator of Dlx1/2 expression in interneuron
progenitors. However, whether the molecular mechanisms underlying this
regulation are direct or indirect was not known. Our results provide the first
evidence for a direct regulation of the Dlx1/2 genes by MASH1. We
have demonstrated the presence of a sequence (E-box) recognized by MASH1 in
the FP5 site of the I12b enhancer. Transgenic embryos generated using a mutant
I12b enhancer (FP5-I12b-lacZ), in which the E-box was
destroyed, displayed an almost complete loss of I12b enhancer activity in the
telencephalon of E11.5 embryos. This result is surprising considering that
Dlx1/2 expression in the telencephalon is not completely abolished in
Mash1-/- embryos
(Casarosa et al., 1999). In
fact, Dlx gene expression in the LGE and MGE of these mutants is upregulated.
This is only an apparent contradiction: it must be remembered that I12b
activity recapitulates only part of the Dlx1/2 expression patterns.
At least one other enhancer, URE2
(Hamilton et al., 2005) (N.G.,
M. Yu, J. Long, G.H., J. L. R. Rubenstein and M.E., unpublished) is also
involved in Dlx1/2 regulation in the forebrain in an overlapping but
distinct population of cells. Thus, in Mash1-null mice,
Dlx1/2 expression can be increased in a cell population in which Dlx
expression is I12b-independent but depends instead, for example, on URE2. This
result can also be explained by the non-cell-autonomous function of
Mash1 in the developing telencephalon. Thus, in Mash1-null
mice, neighboring progenitors could undergo premature differentiation and
express Dlx1/2 in an I12b-independent fashion.
Concluding remarks
The disruption of interneuron development could be linked to
neurodevelopmental disorders (Berretta et
al., 2004; Cobos et al.,
2005; Cossart et al.,
2005; Dykens et al.,
2004; Horike et al.,
2005; Levitt et al.,
2004; Magloczky and Freund,
2005; Rubenstein and
Merzenich, 2003; Samaco et
al., 2005). Interestingly, Dlx genes have been shown to be linked
to epilepsia and Rett syndrome (Cobos et
al., 2005; Horike et al.,
2005). Also, members of the Dlx homeobox gene family are found in
two autism-susceptibility loci, chromosome 2q and 7q
(International Molecular Genetic Study of
Autism Consortium, 2001). Rubenstein and Merzenich recently
proposed a model of autism in which an increased ratio of
excitation/inhibition on a given neuronal network is suggested to cause some
forms of autism (Rubenstein and Merzenich,
2003). Single nucleotide polymorphisms (SNPs) were found at the
Dlx1/2 and Dlx5/6 loci, including in ultraconserved
(Bejerano et al., 2004)
regulatory elements controlling Dlx gene expression in the forebrain (URE2,
I12b, I56i and I56ii) (Hamilton et al.,
2005). The presence of a SNP in these ultraconserved regulatory
elements is not only surprising, but also strongly suggests that the SNP could
be causally related to the behavioral defects seen in the autistic individuals
that carry it. Thus, elucidation of the genetic cascades controlling Dlx gene
expression through these enhancers will enhance our knowledge of GABAergic
interneuron development, as well as providing new insights in the
understanding of important neurological disorders.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1755/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-Mash1). PCR analysis was performed on
the immunoprecipitated chromatin using primers corresponding to the I12b
enhancer (I12b Enh) and the Hes6 upstream enhancer (Hes6 Enh). After
28-30 cycles, PCR products (605 bp for mI12b and 176 bp for Hes6) were
electrophoresed on 1% agarose gel. As controls, amplifications were carried
out on chromatin obtained from the elution of protein-A sepharose beads that
was preincubated with the cross-linked chromatin or with the IP dilution
buffer (No antibody and Mock, respectively). Different dilutions of total
input chromatin (Genomic DNA) were used for each set of primers: 1/500 for
I12b and 1/1000 for Hes6 Enh.