During embryonic development, two major types of neurons
migrate and populate the cortex: the excitatory glutamate-releasing
neurons and the inhibitory -aminobutyric acid (GABA)-releasing
interneurons. It has been proposed that some forms of autism are
the result of an imbalance in the excitation/inhibition ratio of the
cortical neurons, leading to an increase in the excitatory state of the
brain (Rubenstein and Merzenich, 2003). This excitatory state
could be achieved by a reduction in the inhibitory signaling or an
increase in the excitatory system.
The differentiation of multipotent neuronal progenitors into
various neuronal cell types requires complex genetic regulatory
mechanisms. For instance, the differentiation and proper migration
of the GABAergic interneurons to the cortex are controlled to a
large extent by homeodomain-containing transcription factors of
the Dlx family (Anderson et al., 1997a; Anderson et al., 1997b).
Four Dlx genes, Dlx1, Dlx2, Dlx5 and Dlx6, are expressed in two
distinct forebrain domains: the ventral telencephalon and parts of
the diencephalon (Bulfone et al., 1993; Robinson et al., 1991).
Their expression in the ventral telencephalon is restricted to the
differentiating GABAergic projection neurons and interneurons,
which will later migrate to the cortex and olfactory bulb (Anderson
et al., 1997a; Stuhmer et al., 2002a; Stuhmer et al., 2002b).
The Dlx genes are organized in convergently transcribed bigene
clusters and have highly similar patterns of expression, possibly
owing to shared cis-acting regulatory elements (CREs) (Nakamura
et al., 1996; Porteus et al., 1991; Price et al., 1991; Robinson and
Mahon, 1994; Robinson et al., 1991; Scherer et al., 1995; Simeone
et al., 1994; Stock et al., 1996). Through phylogenetic footprinting
and transgenic analysis, two CREs, called I56i and I56ii, have been
discovered in the Dlx5/Dlx6 intergenic region (Zerucha et al.,
2000). In reporter assays, both elements are active in the
telencephalon and diencephalon. In-depth analysis of their
enhancer activities has shown that these individual enhancers may
participate in Dlx gene regulation during the development of
specific populations of GABAergic interneurons (Ghanem et al.,
2007; Ghanem et al., 2008).
The Dlx genes have been associated with human autism
spectrum disorders (ASDs) (Hamilton et al., 2005). Two Dlx
bigene clusters, DLX1/DLX2 and DLX5/DLX6, are found in autism
susceptibility loci on chromosomes 2q31.1 and 7q21.3. Two recent
linkage studies have provided evidence that these two loci can be
linked to ASDs (Liu et al., 2009; Nakashima et al., 2009). Liu and
collaborators have shown that two polymorphisms found in the
DLX1/DLX2 loci could potentially increase susceptibility or cause
autism in cohorts of multiplex families in which there is a greater
genetic component for these conditions (Liu et al., 2009). In a
search for autism genomic variants in the DLX1/2 and DLX5/6 loci,
we identified 31 single-nucleotide polymorphisms (SNPs) and two
insertion/deletion polymorphisms. One adenine-to-guanine SNP
was found at position 182 of I56i (Hamilton et al., 2005). The
location of the SNP coincides with one of the two putative Dlx
Development 137, 3089-3097 (2010) doi:10.1242/dev.051052
© 2010. Published by The Company of Biologists Ltd
1Center for Advanced Research in Environmental Genomics (CAREG), Department of
Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada. 2Centre de Recherche
du CHUQ-CHUL, Faculty of Medicine, Laval University, QC G1V 4G2, Canada.
3Proteomics Platform of the Québec Genomic Center, Centre de Recherche du
CHUQ-CHUL, Laval University, QC G1V 4G2, Canada. 4Department of Psychiatry,
University of California, San Francisco, CA 94158-2324, USA. 5Institute for Human
Genetics, University of California, San Francisco, CA 94158-2324, USA. 6Nina
Ireland Laboratory, University of California, San Francisco, CA 94158-2324, USA.
*Author for correspondence (firstname.lastname@example.org)
Accepted 30 June 2010
Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to
various subtypes of -aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons.
Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy,
schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an
autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is
involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in
reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially
migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP
affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx
proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general
transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a
neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an
ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.
KEY WORDS: Autism, Enhancer, Polymorphism, Telencephalon, Transgenics, Mouse
An SNP in an ultraconserved regulatory element affects
Dlx5/Dlx6 regulation in the forebrain
Luc Poitras1, Man Yu1, Cindy Lesage-Pelletier1, Ryan B. MacDonald1, Jean-Philippe Gagné2, Gary Hatch1,
Isabelle Kelly3, Steven P. Hamilton4,5, John L. R. Rubenstein5,6, Guy G. Poirier2,3and Marc Ekker1,*
binding sites (Feledy et al., 1999) within I56i (Zerucha et al.,
2000). This region of I56i also encodes the second exon of the long
polyadenylated non-coding RNA (lpncRNA) Evf2 (Dlx6os1 –
Mouse Genome Informatics) (Feng et al., 2006), which was
recently shown to contribute to the mechanisms by which the
Dlx5/Dlx6 bigene cluster is regulated (Bond et al., 2009).
Furthermore, the I56i CRE was identified as one of 481
ultraconserved elements of the mouse genome (exhibiting 100%
identity, with no insertion or deletions, on DNA fragments longer
than 200 bp between orthologous regions of the human, rat and
mouse genomes) (Bejerano et al., 2004). The 182-SNP was found
to be part of a completely conserved 8 bp motif based on the
sequence conservation between 40 vertebrate genomes (Fig. 1).
Therefore, the unexpected identification of this SNP in the I56i
enhancer suggested that it could have functional consequences for
Dlx5/Dlx6 regulation during development.
Here, we present evidence suggesting that the I56i SNP affects
transcriptional regulation of the Dlx5/Dlx6 bigene cluster through
the I56i CRE. Enhancer activity is impaired by the I56i SNP,
leading to a decrease in the expression of a reporter gene in the
developing telencephalon as well as in different subpopulations of
GABAergic neurons in the adult cortex. Our in vivo and in vitro
assays strongly suggest that Dlx proteins have a reduced affinity
for the variant site. Affinity purification confirmed that Dlx
proteins are bound at or near the SNP sequence and also led to the
identification of Gtf2i as a new regulator of the Dlx5/Dlx6 bigene
cluster. We propose that the impaired I56i enhancer activity
resulting from the 182-SNP could impact on Dlx function and
therefore cortical interneuron development and could constitute a
contributing factor to developmental abnormalities underlying
MATERIALS AND METHODS
Mutagenesis of mouse I56i
The 182-SNP-containing I56i enhancer was generated by PCR using the
overlapping fragment technique. A first fragment was amplified from the
mouse I56i enhancer using oligonucleotides I56i-1.for and I56i-316.rev
(for oligonucleotide sequences, see Table S1 in the supplementary
material). A second overlapping fragment was generated using an
oligonucleotide containing the desired mutation I56i SNP in combination
with the I56i-436 primer. After purification by agarose gel electrophoresis,
the two fragments were used as templates for a final PCR reaction with
I56i-1.for and I56i-436.rev. A mix (1:3) of Taq and Pfx DNA polymerase
(Invitrogen) was used to avoid unwanted mutations. Finally, the mutant
enhancer was purified on a gel, TA-cloned and sequenced.
The I56i and vI56i enhancers were subcloned into the XhoI site of the
p1230 vector (Yee and Rigby, 1993) that contains a human -globin
minimal promoter and a lacZ reporter gene. Transgenic animals were
produced and analyzed as described (Zerucha et al., 2000). Two transgenic
lines and three primary transgenic embryos were obtained for the vI56i-
lacZ construct. These transgenic embryos were compared with two mouse
transgenic lines obtained with the I56i-lacZ construct (Ghanem et al., 2003;
Ghanem et al., 2007; Zerucha et al., 2000).
Histology and double immunohistochemistry
Fixation of E13.5 telencephalon was carried out overnight in 4%
paraformaldehyde (PFA) in 1?PBS. For adult brains, mice (P30) were
deeply anesthetized and subjected to intracardiac perfusion with 10% saline
solution followed by 4% PFA in 1?PBS. The adult brains were removed
and post-fixed in 4% PFA for 2 hours at room temperature. After fixation,
all brains were cryoprotected by immersion in 30% sucrose, frozen in OCT
(Tissue-Tek) on dry ice, and stored at –80°C until use. For single and
double immunohistochemistry, E13.5 brains were cut at 60 m and adult
brains at 40-45 m on a cryostat (Leica CM3050 S). Double
immunostaining was performed as previously described (Ghanem et al.,
2007). An antibody from Abnova
was used for Gtf2i
Nuclear extract preparation
Nuclear extracts were prepared from the brains of E13.5 mouse embryos
(1 ml of tissue) as described (Sambrook et al., 1989). 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 l
were frozen in liquid nitrogen and stored at –80°C.
DNase I footprinting analysis
A portion of the I56i enhancer corresponding to nucleotides 187-316 was
amplified by PCR using primers zI56i-187 and zI56i-316. PCR fragments
were TA-cloned in the pDrive vector (Qiagen). This fragment was
recovered by digestion with EcoRI and KpnI followed by gel purification
(QIAquick, Qiagen). Directional labeling of each fragment was performed
by 5? end fill using the large fragment of DNA polymerase I (Invitrogen).
Footprinting reactions were carried out as described (Poitras et al., 2007).
Protein-DNA complex purification
Nuclear protein extracts from E13.5 mouse embryos were incubated with
2 g of a double-stranded biotinylated oligonucleotide encompassing the
two Dlx binding sites (see mI56i FP3-FP5.For and .Rev in Table S1 in the
supplementary material) in 1? EMSA binding buffer [7 mM Tris pH 7.5,
81 mM NaCl, 2.75 mM dithiothreitol, 5 mM MgCl2, 0.05% NP40, 1
mg/ml bovine serum albumin, 25 g/ml poly(dI-dC), 10% glycerol] at
room temperature for 60 minutes. After 30 minutes of incubation,
streptavidin-coated agarose (Sigma, S1638) was added to the reaction.
Protein-DNA complexes coupled to the streptavidin-sepharose beads were
washed with 1? EMSA binding buffer. Elution of the protein components
of the complexes was carried out by boiling the reaction mixture in
Laemmli loading buffer (final concentration 2% SDS, 10% glycerol, 5%
2-mercaptoethanol, 0.002% Bromophenol Blue and 0.0625 M Tris-HCl).
Removal of the sepharose beads was achieved by filtering the boiled
reaction through a SPIN-X centrifuge tube filter (Corning).
Sample preparation for mass spectrometry analysis
Eluted proteins were resolved using a 4-12% Criterion XT Bis-Tris
gradient gel (Bio-Rad) and stained with Sypro Ruby (Invitrogen) according
to the manufacturer’s instructions. Images were acquired on a Geliance
CCD-based bioimaging system (PerkinElmer).
Liquid chromatography (LC)-tandem mass spectrometry (MS/MS)
The entire protein profile on SDS-PAGE was sliced from the gel into 25
bands using a gel excision Lanepicker (The Gel Company). In-gel protein
digestion was performed on a MassPrep liquid handling station
(Micromass) according to the manufacturer’s specifications and using
sequencing grade modified trypsin (Promega). Peptide extracts were dried
using a SpeedVac.
Peptide extracts were separated by online reversed-phase (RP) nanoscale
capillary liquid chromatography (nanoLC) and analyzed by electrospray
mass spectrometry (ES MS/MS). The experiments were performed on a
Thermo Surveyor MS pump connected to an LTQ linear ion-trap mass
spectrometer equipped with a nanoelectrospray ion source (Thermo
Electron, San Jose, CA, USA). Peptide separation took place within a
PicoFrit BioBasic C18 column of 10 cm ? 0.075 mm internal diameter
(New Objective, Woburn, MA, USA) with a linear gradient from 2 to 50%
solvent B (acetonitrile, 0.1% formic acid) in 30 minutes at 200 nl/minute.
Mass spectra were acquired using the data-dependent acquisition mode
(Xcalibure Software, version 2.0). Each full-scan mass spectrum (400 to
2000 m/z) was followed by collision-induced dissociation of the seven
most intense ions. The dynamic exclusion function was enabled (30 second
exclusion) and the relative collisional fragmentation energy was set to 35%.
Development 137 (18)
Feledy, J. A., Morasso, M. I., Jang, S.-I. and Sargent, T. D. (1999).
Transcriptional activation by the homeodomain protein Distal-less 3. Nucl Acids
Res. 27, 764-770.
Feng, J., Bi, C., Clark, B. S., Mady, R., Shah, P. and Kohtz, J. D. (2006). The Evf-
2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and
functions as a Dlx-2 transcriptional coactivator. Genes Dev. 20, 1470-1484.
Fogarty, M., Grist, M., Gelman, D., Marín, O., Pachnis, V. and Kessaris, N.
(2007). Spatial genetic patterning of the embryonic neuroepithelium generates
GABAergic interneuron diversity in the adult cortex. J. Neurosci. 27, 10935-10946.
Francke, U. (1999). Williams-Beuren syndrome: genes and mechanisms. Hum.
Mol. Genet. 8, 1947-1954.
Ghanem, N., Jarinova, O., Amores, A., Hatch, G., Park, B. K., Rubenstein, J.
L. R. and Ekker, M. (2003). Regulatory roles of conserved intergenic domains in
vertebrate Dlx bigene clusters. Genome Res. 13, 533-543.
Ghanem, N., Yu, M., Long, J., Hatch, G., Rubenstein, J. L. and Ekker, M.
(2007). Distinct cis-regulatory elements from the Dlx1/Dlx2 locus mark different
progenitor cell populations in the ganglionic eminences and different subtypes
of adult cortical interneurons. J. Neurosci. 27, 5012-5022.
Ghanem, N., Yu, M., Poitras, L., Rubenstein, J. and Ekker, M. (2008).
Characterization of a distinct subpopulation of striatal projection neurons
expressing the Dlx genes in the basal ganglia through the activity of the I56ii
enhancer. Dev. Biol. 322, 415-424.
Hamilton, S. P., Woo, J. M., Carlson, E. J., Ghanem, N., Ekker, M. and
Rubenstein, J. L. (2005). Analysis of four DLX homeobox genes in autistic
probands. BMC Genet. 6, 52.
Horike, S., Cai, S., Miyano, M., Cheng, J. F. and Kohwi-Shigematsu, T. (2005).
Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett
syndrome. Nat. Genet. 37, 31-40.
Keller, A., Nesvizhskii, A., Kolker, E. and Aebersold, R. (2002). Empirical
statistical model to estimate the accuracy of peptide identifications made by
MS/MS and database search. Anal. Chem. 74, 5383-5392.
Kimura, M., Kazuki, Y., Kashiwagi, A., Kai, Y., Abe, S., Barbieri, O., Levi, G.
and Oshimura, M. (2004). Dlx5, the mouse homologue of the human-
imprinted DLX5 gene, is biallelically expressed in the mouse brain. J. Hum.
Genet. 49, 273-277.
Klein-Tasman, B., Mervis, C., Lord, C. and Phillips, K. (2007). Socio-
communicative deficits in young children with Williams syndrome: performance on
the Autism Diagnostic Observation Schedule. Child Neuropsychol. 13, 444-467.
Le T., Du, G., Fonseca, M., Zhou, Q., Wigle, J. and Eisenstat, D. (2007). Dlx
homeobox genes promote cortical interneuron migration from the basal
forebrain by direct repression of the semaphorin receptor neuropilin-2. J. Biol.
Chem. 282, 19071-19081.
Lettice, L., Hill, A., Devenney, P. and Hill, R. (2008). Point mutations in a distant
sonic hedgehog cis-regulator generate a variable regulatory output responsible
for preaxial polydactyly. Hum. Mol. Genet. 17, 978-985.
Lincoln, A., Searcy, Y., Jones, W. and Lord, C. (2007). Social interaction
behaviors discriminate young children with autism and Williams syndrome. J.
Am. Acad. Child Adolesc. Psychiatry 46, 323-331.
Liu, X., Novosedlik, N., Wang, A., Hudson, M., Cohen, I., Chudley, A.,
Forster-Gibson, C., Lewis, S. and Holden, J. (2009). The DLX1and DLX2
genes and susceptibility to autism spectrum disorders. Eur. J. Hum. Genet. 17,
Long, J., Cobos, I., Potter, G. and Rubenstein, J. (2009a). Dlx1&2 and Mash1
transcription factors control MGE and CGE patterning and differentiation
through parallel and overlapping pathways. Cereb. Cortex 19, i96-i106.
Long, J., Swan, C., Liang, W., Cobos, I., Potter, G. and Rubenstein, J. (2009b).
Dlx1&2 and Mash1 transcription factors control striatal patterning and
differentiation through parallel and overlapping pathways. J. Comp. Neurol.
Nakamura, S., Stock, D. W., Wynder, K. L., Bollekens, J. A., Takeshita, K.,
Nagai, B. M., Chiba, S., Kitamura, T., Freeland, T. M., Zhao, Z. et al. (1996).
Genomic analysis of a new mammalian distal-less gene: Dlx7. Genomics 38,
Nakashima, N., Yamagata, T., Mori, M., Kuwajima, M., Suwa, K. and
Momoi, M. (2009). Expression analysis and mutation detection of DLX5 and
DLX6 in autism. Brain Dev. 32, 98-104.
Nesvizhskii, A., Keller, A., Kolker, E. and Aebersold, R. (2003). A statistical
model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75,
Okita, C., Meguro, M., Hoshiya, H., Haruta, M., Sakamoto, Y. K. and
Oshimura, M. (2003). A new imprinted cluster on the human chromosome
7q21-q31, identified by human-mouse monochromosomal hybrids. Genomics
Poitras, L., Ghanem, N., Hatch, G. and Ekker, M. (2007). The proneural
determinant MASH1 regulates forebrain Dlx1/2 expression through the I12b
intergenic enhancer. Development 134, 1755-1765.
Porteus, M. H., Bulfone, A., Ciaranello, R. D. and Rubenstein, J. L. R. (1991).
Isolation and characterization of a novel cDNA clone encoding a homeodomain
that is developmentally regulated in the ventral forebrain. Neuron 7, 221-229.
Price, M., Lemaistre, M., Pischetola, M., Di Lauro, R. and Duboule, D. (1991).
A mouse gene related to distal-less shows a restricted expression in the
developing forebrain. Nature 351, 748-751.
Richler, E., Reichert, J. G., Buxbaum, J. D. and McInnes, L. A. (2006). Autism
and ultraconserved non-coding sequence on chromosome 7q. Psychiatr. Genet.
Robinson, G. W. and Mahon, K. A. (1994). Differential and overlapping
expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less
homeobox genes in craniofacial development. Mech. Dev. 48, 199-215.
Robinson, G. W., Wray, S. and Mahon, K. A. (1991). Spatially restricted
expression of a member of a new family of murine distal-less homeobox genes
in the developing forebrain. New Biol. 3, 1183-1194.
Rubenstein, J. L. and Merzenich, M. M. (2003). Model of autism: increased
ratio of excitation/inhibition in key neural systems. Genes Brain Behav. 2, 255-
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning, a
Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor
Scherer, S. W., Heng, H. H. Q., Robinson, G. W., Mahon, K. A., Evans, J. P.
and Tsui, L.-C. (1995). Assignment of the human homolog of mouse Dlx3 to
chromosome 17q21.3-q22 by analysis of somatic cell hybrids and fluorescence
in situ hybridization. Mamm. Genome 6, 310-311.
Schule, B., Li, H. H., Fisch-Kohl, C., Purmann, C. and Francke, U. (2007). DLX5
and DLX6 expression is biallelic and not modulated by MeCP2 deficiency. Am. J.
Hum. Genet. 81, 492-506.
Simeone, A., Acampora, D., Pannese, M., Desposito, M., Stornaiuolo, A.,
Gulisano, M., Mallamaci, A., Kastury, K., Druck, T., Huebner, K. et al.
(1994). Cloning and characterization of two members of the vertebrate Dlx gene
family. Proc. Natl. Acad. Sci. USA 91, 2250-2254.
Skuse, D. H. (2007). Rethinking the nature of genetic vulnerability to autistic
spectrum disorders. Trends Genet. 23, 387-395.
Stock, D. W., Ellies, D. L., Zhao, Z., Ekker, M., Ruddle, F. H. and Weiss, K. M.
(1996). The evolution of the vertebrate Dlx gene family. Proc. Natl. Acad. Sci.
USA 93, 10858-10863.
Stuhmer, T., Anderson, S. A., Ekker, M. and Rubenstein, J. L. R. (2002a).
Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx
expression. Development 129, 245-252.
Stuhmer, T., Puelles, L., Ekker, M. and Rubenstein, J. L. (2002b). Expression
from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons.
Cereb. Cortex 12, 75-85.
Van der Aa, N., Rooms, L., Vandeweyer, G., van den Ende, J., Reyniers, E.,
Fichera, M., Romano, C., Delle Chiaie, B., Mortier, G., Menten, B. et al.
(2009). Fourteen new cases contribute to the characterization of the 7q11.23
microduplication syndrome. Eur. J. Med. Genet. 52, 94-100.
Wang, Y., Dye, C., Sohal, V., Long, J., Estrada, R., Roztocil, T., Lufkin, T.,
Deisseroth, K., Baraban, S. and Rubenstein, J. (2010). Dlx5 and Dlx6
regulate the development of parvalbumin-expressing cortical interneurons. J.
Neurosci. 30, 5334-5345.
Wonders, C. and Anderson, S. (2006). The origin and specification of cortical
interneurons. Nat. Rev. Neurosci. 7, 687-696.
Wonders, C., Taylor, L., Welagen, J., Mbata, I., Xiang, J. and Anderson, S.
(2008). A spatial bias for the origins of interneuron subgroups within the medial
ganglionic eminence. Dev. Biol. 314, 127-136.
Yee, S.-P. and Rigby, P. W. J. (1993). The regulation of myogenin gene expression
during the embryonic development of the mouse. Genes Dev. 7, 1277-1289.
Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu, G., Gambarotta,
A., Schultz, J. R., Rubenstein, J. L. R. and Ekker, M. (2000). A highly
conserved enhancer in the Dlx5/Dlx6 intergenic region is the site of cross-
regulatory interactions between Dlx genes in the embryonic forebrain. J.
Neurosci. 20, 709-721.
Zhang, H., Hu, G., Wang, H., Sciavolino, P., Iler, N., Shen, M. M. and Abate-
Shen, C. (1997). Heterodimerization of Msx and Dlx homeoproteins results in
functional antagonism. Mol. Cell. Biol. 17, 2920-2932.
Zhou, Q. P., Le T. N., Qiu, X., Spencer, V., de Melo, J., Du, G., Plews, M.,
Fonseca, M., Sun, J. M., Davie, J. R. et al. (2004). Identification of a direct Dlx
homeodomain target in the developing mouse forebrain and retina by
optimization of chromatin immunoprecipitation. Nucleic Acids Res. 32, 884-892.
SNP in a Dlx5/6 enhancer