Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons

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Molecular Brain Research 122 (2004) 35–46

Research report

Neuroanatomical distribution of ARX in brain and its localisation

in GABAergic neurons

Karine Poiriera,1, Hilde Van Escha,1, Gaelle Friocourta, Yoann Sailloura, Nadia Bahia,Stephanie Backerb, Evelyne Souilc, Laetitia Castelnau-Ptakhinea, Cherif Beldjorda,

Fiona Francisa, Thierry Bienvenua, Jamel Chellya,*

aLaboratoire de Genetique et de Physiopathologie des Retards Mentaux, Institut Cochin. Inserm U567, Universite Paris V. 24,

rue du Faubourg Saint Jacques, 75014 Paris, FrancebDeveloppement et migration neuronale, Institut Cochin. Inserm U567, Universite Paris V. 24, rue du Faubourg Saint Jacques, 75014 Paris, France

cPlateforme de Morphologie et Histologie du petit animal, Institut Cochin, CNRS, INSERM, Universite Paris V. 123,

Boulevard Port-Royal, 75014 Paris, France

Accepted 11 November 2003

Abstract

Recent human genetics approaches identified the Aristaless-related homeobox (ARX) gene as the causative gene in X-linked infantile

spasms, Partington syndrome, and non-syndromic mental retardation as well as in forms of lissencephaly with abnormal genitalia. The ARX

predicted protein belongs to a large family of homeoproteins and is characterised by a C-terminal Aristaless domain and an octapeptide

domain near the N-terminus. In order to learn more about ARX function, we have studied in detail Arx expression in the central nervous

system during mouse embryonic development as well as in the adult. During early stages of development, Arx is expressed in a significant

proportion of neurons in the cortex, the striatum, the ganglionic eminences and also in the spinal cord. In the adult, expression of Arx is still

present and restricted to regions that are known to be rich in GABAergic neurons such as the amygdala and the olfactory bulb. A possible role

for Arx in this type of neurons is further reinforced by the expression of Arx in a subset of GABAergic interneurons in young and mature

primary cultures of cortical neuronal cells as well as in vivo. Moreover, these data could explain the occurrence of seizures in the great

majority of patients with an ARX mutation, due to mislocalisation or dysfunction of GABAergic neurons. We also performed ARX wild-type

and mutant over-expression experiments and found that the different ARX mutations tested did not modify the morphology of the cells.

Moreover, no abnormal cell death or protein aggregation was observed, hence suggesting that more subtle pathogenic mechanisms are

involved.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Developmental disorders

Keywords: ARX; Mental retardation; Brain development; GABA; Mouse

1. Introduction cluding patients with dystonia, and patients with myoclonic

The human Aristaless-related homeobox gene (ARX)

was identified as the causative gene in X-linked infantile

spasms (ISSX; West syndrome, MIM308350), in certain

families with X-linked syndromic (Partington syndrome,

MIM309510) and non-syndromic mental retardation, in-

0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.molbrainres.2003.11.021

* Corresponding author. Tel.: +33-1-44-41-24-81; fax: +33-1-44-21-

24-21.

E-mail address: [email protected] (J. Chelly).1 These authors contirbuted equally to this work.

epilepsy with spasticity [5,20]. Mutations identified in ARX

in these patients included missense mutations, a recurrent

in-frame 24-bp duplication predicted to cause an expansion

of a polyalanine tract from 12 to 20 alanines and a small in-

frame insertion leading to an increase of another polyala-

nine tract containing 16 residues. Notably, no nonsense

mutations were identified in these cases. More recently,

mutations in ARX have been identified in X-linked lissen-

cephaly with abnormal genitalia (XLAG, MIM300215) [9].

However, these latter mutations in ARX result predominant-

ly in premature termination signals because of frameshifts

K. Poirier et al. / Molecular Brain Research 122 (2004) 35–4636

due to insertions or deletions, or nonsense mutations. In

two cases of XLAG, missense mutations have been iden-

tified (R332H, L343Q) [9]. These two mutations are

located in highly conserved regions of the homeodomain

suggesting dysfunction of DNA binding. The striking

differences in phenotype, which are observed (XLAG vs.

X-linked mental retardation), are likely to be explained by

the consequences of the different ARX mutations on the

stability and/or the function of ARX protein.

The ARX predicted protein belongs to one of the three

largest classes of homeoproteins, the paired (Prd) class.

Within this class, it is member of a specific sub-class of

proteins, which contain a glutamine residue at the critical

position 50 of their homeodomain (Q50), a residue also

found conserved in the Drosophila Aristaless protein (al).

In addition to its paired/Q50 central homeodomain, ARX is

characterised by a 14 amino acid C-terminal Aristaless

domain and by an octapeptide domain located near the

N-terminus designated as the Goosecoid Engrailed Homol-

ogy (GEH) or the eh-1 domain in the Engrailed (En)

homeoprotein [11].

In order to better understand the function of ARX and its

involvement in X-linked mental retardation, we studied Arx

expression in the central nervous system (CNS) during

mouse embryonic development as well as in the adult.

Our results suggest that Arx is likely to play an important

role during embryonic CNS development, as well as in the

adult were ARX expression is still present in the neocortex,

hippocampus, hypothalamus, amygdala and especially in

the olfactory bulb. High expression of Arx in regions which

are known to be rich in GABAergic neurons, and the

expression of Arx in a subset of GABAergic neurons

suggest that mutations in ARX alter GABAergic neuronal

development and/or function, which might contribute to the

seizure phenotype observed in ARX-related disorders.

Over-expression of wild-type and different ARX mutants

involved in certain of these disorders, including a polyala-

nine tract expansion, revealed no dramatic changes in cell

differentiation, morphology and cell death. Moreover, no

abnormal protein aggregation was observed, suggesting that

these mutations result in more subtle cellular changes.

2. Materials and methods

2.1. Expression constructs

The QuikChangek Site-Directed Mutagenesis Kit (Stra-

tagene, La Jolla, CA, USA) was used to introduce the L33P

and the P353L mutations into the mouse Arx cDNA,

subcloned in the pEGFP-C1 vector (Clontech, Palo Alto,

CA, USA). All procedures were performed according to the

manufacturer’s instructions. In the case of the polyalanine

stretch expansion construct (ARX-polyA), we amplified

exon 2 of a patient known to carry the 7 alanine insertion

within the first polyalanine stretch. This exon was subse-

quently subcloned in the mouse Arx cDNA. A final expan-

sion from 16 to 25 alanines within the first polyalanine

stretch of the construct was obtained. All constructs were

confirmed by DNA sequencing (ABI 373, Applied Biosys-

tems, Foster City, CA, USA).

2.2. Antibodies

Two GST-fusion proteins corresponding to amino acids

(aa) 310–396 and aa 496–562 were used to produce

polyclonal antibodies (CovalAb, Oullins, France) against

the Arx homeodomain (anti-ARX-HD) and C-ter domain

(anti-ARX-Cter), respectively. Bleeds were taken after 10

weeks and tested on cell lysates. For Western blot analysis,

anti-Arx antibodies and anti-actin mouse monoclonal anti-

bodies (Chemicon, Temacula, CA, USA) were used at

1:1000. Anti-ARX-antibodies were used at 1:1000 for

immunohistochemistry and 1:100 for immunocytochemis-

try. Anti-a-tubulin (Sigma, 1:2000), anti-h-III tubulin

(BAbCo, Richmond, CA, USA, 1:1000), anti-CaMKII (Sig-

ma, 1:200) and anti-GABA (Sigma, 1:500) mouse mono-

clonal antibodies were also used for immunocytochemistry.

Specific immunostaining using anti-GABA and anti-CaM-

KII mouse monoclonal antibodies was observed only with

rat tissues and primary cultures of neuronal cells.

2.3. GST-fusion proteins production

Two GST-fusion proteins corresponding to the Arx

homeodomain (aa 310–396; GST-HD) and C-ter domain

(aa 496–562; GST-Cter) were produced in B27 bacteria

using IPTG induction. Protein purification was then per-

formed using glutathione Sepharose 4B (Amersham Phar-

macia, Uppsala, Sweden).

2.4. Cell culture and transfection

Animal handling and experiments were performed in

accordance with French ministry of Agriculture recommen-

dations and EEC regulations and guidelines. Adult pregnant

female rats were sacrificed by cervical dislocation and

brains were extracted from day 18 (E18) rat embryos in

phosphate-buffered saline (PBS) containing 0.6% glucose.

After a mechanic dissociation of cortex, living neuronal

cells were counted using trypan blue and cultured in neuro-

basal medium supplemented with 2% B-27 (Invitrogen,

Groningen, The Netherlands) and 100 unit/ml of penicillin

and streptomycin each, until fixation. Neurons were cultured

on glass coverslips or Petri dishes, coated with poly-D-lysine

(Invitrogen) and laminin (Sigma) [4]. Primary glial cultures

were prepared as previously described, from newborn cere-

bral hemispheres [16]. Glial cells were cultured in Dulbec-

co’s modified Eagle’s medium (DMEM, Invitrogen)

supplemented with 4.5 mg/ml glucose, 10% foetal bovine

serum (Invitrogen) and 100 units/ml of penicillin and

streptomycin each. PC12 cells were grown in DMEM

K. Poirier et al. / Molecular Brain Research 122 (2004) 35–46 37

supplemented with glucose (4.5 mg/ml), 5% foetal bovine

serum, 10% horse serum, and 100 units/ml of penicillin and

streptomycin. Plasmid constructs were introduced into PC12

cells (80% confluent) using GenePorter (Gene Therapy

Systems, San Diego, CA, USA) according to the manufac-

turer’s instruction. COS7 cells were cultured in DMEM

supplemented with glucose (4.5 mg/ml), 10% foetal bovine

serum, and 100 units/ml of penicillin and streptomycin each.

Cells were plated at 106 cells per 10-cm culture dishes and

allowed 24 h to adhere before they were transfected using

FuGENE 6 transfection reagent (Roche Molecular Bio-

chemicals, Basel, Switzerland). Expression was allowed

for 24 h before cells were used for immunofluorescence

and biochemical studies. Transfection of PC12 and COS7

cells with all the different constructs was repeated three

times and each time approximately 75 transfected cells were

analysed.

2.5. Immunofluorescence staining and

immunohistochemistry

For immunostaining, PC12 cells were grown on poly-D-

lysine-coated glass coverslips. Immunostaining experiments

using neuronal cells were performed at 5, 10 or 14 days in

culture. COS7, PC12, glial and neuronal cells were fixed in

4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) for 30

min at room temperature. After three washes with 0.1 M

PBS, fixed cells were incubated 1 h in TBST (10 mM Tris–

HCl pH8, 150 mM NaCl, 0.05% Triton X-100) containing

2% goat serum (Dako, Glostrup, Denmark) and then incu-

bated overnight at 4 jC with the primary antibody. The cells

Fig. 1. Specificity of anti-Arx antibody (anti-ARX-HD). (A) Specificity of anti-AR

kDa was observed in E17 rat brain embryo and neonatal brain (NB) extracts. Not

assay. In lanes 1 and 2, equal amounts of adult olfactory bulb protein extracts we

lane 1 was not detected in lane 2 where the antibody was pre-incubated with specifi

transfected COS7 protein extracts. (C) Specificity of anti-ARX-HD tested using

transfected with ARX-GFP, while in lanes 1 and 3 GFP alone was used. The same b

the anti-ARX-HD and the anti-GFP antibodies in lanes 2 and 4.

were washed three times in TBST and incubated for 1 h at

room temperature with affinity purified secondary antibod-

ies (FITC conjugated goat anti rabbit or Texas Red conju-

gated goat anti rabbit antibodies (Jackson Immunoresearch,

West Grove, PA, USA) used at 1:200 dilution. After three

washes, coverslips were mounted in Mowiol and examined

with a Zeiss microscope equipped with epifluorescence

illumination.

Mouse and rat embryos, newborn (P4) and adult brains

were cryoprotected immediately after dissection with 30%

sucrose in phosphate buffer. Embryos were prefixed by

immersion in 2% PFA before being frozen with isopentane

and sectioned (10–15 Am thick). Embryos, newborn and

adult brains sections were then fixed for 20 min (embryos in

2% PFA, newborn and adult brains in 4% PFA) and they

were washed three times with PBS. Immunostaining and

analysis of the sections were performed as described previ-

ously. All immunostaining experiments were performed

with both anti-ARX-antibodies.

2.6. Immunoperoxidase labeling

ARX immunoperoxidase labeling was performed on

free-floating sections of 50 Am obtained from E13.5 mouse

after fixation overnight in 4% paraformaldehyde (PFA).

Endogenous peroxidase activity was blocked by a 30-min

treatment in 0.3% hydrogen peroxidase in phosphate-buff-

ered saline. Sections were pre-incubated in 10% goat serum

and 0.3% Triton X-100 in PBS for an hour and then

incubated with anti-ARX (1/200) in PBS overnight at 4

jC. After three PBS washes, sections were incubated with

X-HD for detecting endogenous Arx. A specific band of approximately 70

e the absence of this protein in cerebellum (Cb). (B) Antibody competition

re loaded on an SDS-PAGE gel. The specific band corresponding to Arx in

c GST-HD fusion protein. Lanes 3 and 4 show the same result obtained with

transfected COS7 cell extracts. Lanes 2 and 4 contain extracts from cells

ands, corresponding to the ARX-GFP fusion protein, were detected by both


the secondary antibody for 2 h at room temperature (bio-

tinylated goat anti-rabbit IgG, Vector Laboratories, Burlin-

game, CA). They were then transferred into avidin–biotin–

peroxydase complex (ABC kit, Vector Laboratories) for 90

min at room temperature. Peroxidase enzyme activity was

revealed using 3,3V-diaminobenzidinetetrahydrochloride

(DAB, final concentration 0.005%) as the chromogen and

0.01% hydrogen peroxide as the substrate. The sections

were then mounted onto gelatine-coated slides, dehydrated

and coverslipped.

2.7. Protein extracts and western immunoblot

Proteins from dissected adult mouse brains were extracted

in lysis buffer (50 mM Tris, 150 mMNaCl, 1% NP40 pH8.0)

Fig. 2. Expression of Arx during mouse embryonic CNS development. (A) Coro

labelling) or black (peroxidase labelling) (b,d,e) and for h� III tubulin in red (a). a

in the ganglionic eminences from where it expanded toward the developing neoc

routes followed by MGE-derived interneurons (c). d–e: By peroxidase immuno

preplate and VZ cells (d,e). CX: cortex; LGE: lateral ganglionic eminence; MGE

ventricular zone. (B) E16.5 coronal sections of neocortex stained for Arx in green

expression was predominantly seen in the marginal zone and in neurons migrating t

also present in the ventricular zone (i). Higher magnifications of the marginal zone

Retzius cells (q). CP: cortical plate; IZ: intermediate zone; MZ: marginal zone; P

on ice. Subsequently, the protein extracts were resuspended

in Laemmli sample buffer (BioRad, Richmond, CA, USA)

and heated at 100 jC for 7 min prior to separation on SDS-

polyacrylamide gels. Transfer to nitro-cellulose membranes

and immunoblotting were carried out following standard

protocols.

3. Results

3.1. Characterisation of polyclonal antibodies specific to

Arx

Two different polyclonal antibodies, one directed against

the C-terminal part of the protein (anti-ARX-Cter) and the

nal and sagittal E13.5 brain sections stained for Arx in green (fluorescent

–c: A strong expression of Arx was present in the developing striatum and

ortex (pre-plate and ventricular zone). Arrows indicate the main migration

staining, Arx expression in the cortex is localised in the nucleus of some

: medial ganglionic eminence; PP: preplate; ST: developing striatum; VZ:

(h,l,p), for Reelin (g,k,o) in red and DAPI in blue (f,j,n). f–q: At E16.5, Arx

hrough the intermediate zone and the cortical plate. In addition, staining was

(20X-63X, j–q) show no apparent co-expression of Arx and Reelin in Cajal-

P: pre-plate; SP: sub-plate; VZ: ventricular zone.

Fig. 2 (continued).


other against the homeodomain (anti-ARX-HD), were used

to detect the Arx protein. As shown in Fig. 1A, anti-ARX-

HD antibody revealed a band of approximately 70 kDa in

rat neonatal and embryo (E17) brain extracts. In protein

extracts from cerebellum, no protein was detected with these

antibodies. This result is in line with the previously de-

scribed in situ hybridisation and RT-PCR expression studies

that showed an absence of ARX mRNA expression in the

cerebellum [5]. The detected protein is likely to be specific

and corresponds to the native ARX protein, since pre-

immune sera did not produce a signal on the Western blots

and competitive assay of the antibodies with the specific

GST-fusion protein displaced completely the detected band

(Fig. 1B). Moreover, a similar band corresponding to an

ARX-GFP fusion protein obtained by transfection in COS7

cells was recognised by both anti-ARX-HD and anti-GFP

antibodies (Fig. 1C). Similar results were obtained for anti-

ARX-Cter (data not shown). The specificity of these anti-

bodies is further supported by the coherent immunostaining

results obtained with both antibodies. In this study, figures

illustrating the reported results were obtained with the anti-

ARX-HD.

3.2. Arx is predominantly expressed in the marginal zone of

the developing neocortex

Previous RNA in situ analysis showed that Arx can be

detected from day E8 onwards [5]. In this report, we

Fig. 3. Expression of Arx in E13.5 mouse spinal cord and pons. E13.5

transverse sections through mouse cervical spinal cord (b) and pons (a)

stained for Arx by immunoperoxidase technique. At E13.5, detailed

transverse sections showed expression of Arx in the area of the pons (a) and

in the floorplate (a–b). In addition, expression of Arx was seen in the

intermediate region of the spinal cord, extending towards the prospective

dorsal horn. (b) D: dorsal; FP: floorplate; V: ventral.


examined the expression of Arx protein in mouse embry-

onic neural tissues including brain and spinal cord by

immunostaining. At E10, a restricted expression of Arx

was observed in the telencephalic vesicles, the ventral

thalamus and in the floor plate (data not shown). At later

stages of development, from E10.5 to E16.5, Arx expres-

sion was observed in telencephalic structures including the

ganglionic eminences, the developing neocortex and the

striatum, in the ventral thalamus (Fig. 2A and B), as well as

in the pons (Fig. 3a) and the medulla oblongata (data not

shown). We did not detect any expression in the rest of the

developing diencephalon neither in the cerebellum (data not

shown). These results are consistent with our previous in

situ hybridisation experiments. At E13.5, a strong expres-

sion of Arx was observed in the developing striatum and in

the intermediate zone of the ganglionic eminences, a region

where interneurons originate [1,10] (Fig. 2A). From this

region, Arx expression expanded toward the developing

neocortex where it was mainly detected in the pre-plate,

characterised by expression of h-III tubulin, a marker of

post-mitotic neurons (Fig. 2A). The number of ARX

positive cells in the pre-plate layer adjacent to the MGE

appears to be much higher than in the preplate

corresponding to a more anterior region of the developing

telencephalon. These results suggest that ARX positive cells

display an anterior–posterior gradient coherent with gradi-

ent of migrating neurons from the MGE to the telenceph-

alon (data not shown). We also observed a staining in the

ventricular zone, suggesting that Arx is expressed in both

dividing and post-mitotic cells (Fig. 2B). The nuclear

localisation of ARX in cells localised in the VZ was

confirmed by peroxidase immunostaining (Fig. 2A, d and

e). At E16.5, Arx expression was predominantly seen in the

marginal zone and in neurons migrating through the inter-

mediate zone and the cortical plate (Fig. 2B). In addition,

staining was still present in the ventricular zone. To further

characterise the type of neurons that expressed Arx in the

marginal zone, we performed co-labelling with Reelin, a

glycoprotein secreted by Cajal-Retzius cells. We did not

observe co-expression of Reelin and Arx in Cajal-Retzius

cells, suggesting that Arx labels another sub-type of early

generated neurons (Fig. 2B).

3.3. Arx expression in the developing spinal cord

We also examined Arx expression in the developing

and adult spinal cord at the cervical level. The expression

of Arx was first seen in the neuroepithelium at E10.5 (data

not shown), confirming our previous in situ data. At this

early stage of development, spinal cord cells are beginning

to differentiate, moving from the ventricular zone to the

intermediate zone during the process of migration. At

E13.5, we observed that Arx was still expressed in the

floorplate and in the area of the pons (Fig. 3). In addition,

an expression was seen in a low proportion of cells in the

mantle layer of the intermediate region and the prospective

dorsal horn (Fig. 3). Analysis at later stages of develop-

ment showed a decreasing number of cells expressing

ARX. In the adult spinal cord, Arx expression is no longer

detected (data not shown).

3.4. Arx expression in the neocortex and in the olfactory

bulb in the adult

We examined the expression of the Arx protein in

adult mouse cortex, striatum, thalamus, colliculi, cerebel-

lum, hippocampus and olfactory bulb by Western blot

analysis using both anti-ARX antibodies. Besides a

strong predominant expression in the olfactory bulb, a

specific band of approximately 70 kDa was present in

the cortex, the striatum, and the thalamus. In the colliculi

and the hippocampus, the Arx protein band was barely

detectable (data not shown). As previously observed by


RNA in situ hybridisation, no expression was detected in

the cerebellum.

To confirm this restricted pattern of Arx expression in the

adult CNS, we performed immunostaining on adult mouse

and rat brain sections. This confirmed that Arx was still

expressed in single cells dispersed in all layers of the

neocortex, in the ventral hypothalamus, in the dentate gyrus

Fig. 4. Expression of Arx in the adult mouse brain. (A) Coronal section through th

Left, a histological section of the mouse brain is given to indicate the different struc

nuclear localisation of ARX in cortical cells is shown with a higher magnification

Granular cell layer, dentate gyrus; PoDG: Polymorph cell layer, dentate gyrus. A

through the olfactory bulb, stained for Arx in green (e) and DAPI in blue (j). Left,

cell layers. Arx expression is evident in glomerular (GL), granule (GR) and mitr

(Fig. 4A) and in a large number of cells in the amygdala

(data not shown) and the olfactory bulb (Fig. 4B). In the

latter, Arx labelled neurons in the granule, glomerular and

mitral cell layers (Fig. 4B). Similar to the results on

embryonic brain, we did not find Arx expression in the

cerebellum. Same results were obtained on adult rat brain

sections (data not shown).

e adult mouse brain, stained for Arx in green (a–d) and DAPI in blue (f– i).

tures where Arx expression was seen. Merged images are shown (k–n). The

(d–n). CX: cortex; DG: dentate gyrus; VH: ventral hypothalamus; GrDG:

ll layers of the neocortex contain Arx positive cells. (B) Coronal section

a histological section of the olfactory bulb is given to indicate the different

al (MI) cell layers.

Brain Research 122 (2004) 35–46

3.5. Arx expression in GABAergic neurons

In order to explore Arx expression in young and matur-

ing neurons, cell extracts from primary cultures of E18 rat

cortical brain neurons grown for 2, 5, 10, 14, 21 and 30 days

(D) were screened with anti-ARX-HD. Expression is al-

ready present in immature neurons at D2 and persisted for

30 days in culture (Fig. 5A). Hence, Arx is detected not only

in young differentiating neurons, but also in mature neurons

K. Poirier et al. / Molecular42

Fig. 5. Arx is predominantly expressed in GABAergic neurons in culture and in

grown for 2, 5, 10, 14, 21 and 30 days, respectively. Arx was found to be expre

double band was detected probably corresponding to post-translation modification

with the protein produced in vitro (data not shown). Anti-actin was used as contro

with a-tubulin (a), CaMKII (e) or GABA (h) in red, DAPI (c) in blue and Arx (b,f

subset of neurons (c versus d). Co-expression was observed in a subset of GA

(C) Coronal sections through the adult rat neocortex showing layers II– IVof the m

red. Merged images (c,g) and higher magnifications (63� , d,h) show that the large

was observed for Arx and CaMKII. The arrow indicates a cell that co-expresse

observed in all parts and layers of the neocortex.

in culture. Neuronal cell cultures of embryonic rat cortex

stained for Arx at D14 of culture, revealed a cell-specific

expression pattern, i.e. only a subset of neuronal cells

express Arx. On the contrary, no Arx expression was

observed in glial cells positive for glial fibrillary acidic

protein (GFAP), a specific marker of this type of cells (data

not shown).

To further characterise the neuronal subset that expressed

Arx, we assessed the expression of calcium-calmodulin-

vivo. (A) Arx expression in primary cultures of cortical rat brain neurons

ssed in both immature and mature neurons in these cultures. In all lanes, a

of the Arx protein or the existence of isoforms. This doublet was also seen

l. (B) Primary neuronal cell cultures of embryonic rat cortex at D14 stained

,i) antibodies in green. Merged images show that Arx is expressed in only a

BAergic neurons (j), while no co-expression was seen with CaMKII (g).

edial cortex, stained for Arx in green (b,f) and GABA (a) and CaMKII (e) in

majority of Arx positive cells co-expresses GABA, while no co-expression

s ARX and GABA. Similar co-expressions of Arx and GABA have been

Fig. 5 (continued).


dependent kinase II (CaMKII) and gamma-aminobutyric acid

(GABA) in young and mature cultures of primary neuronal

cells (cultures of 5, 10 and 14 days). Double immunofluo-

rescent staining using adequate antibodies showed the pres-

ence of dot-like signals for Arx in the nucleus of many cells

that were positive for GABA (Fig. 5B). However, cells

expressing CaMKII, a marker of excitatory glutamatergic

neurons did not express Arx. This pattern of expression was

observed in young as well as in mature cultures. To confirm

this cell-specific expression pattern in vivo, we performed

double-staining experiments on newborn and adult rat brain

sections. GABA/Arx double immunostaining showed that

ARX is expressed in a high proportion of GABAergic cells.

In some brain structures known to be very rich in GABAergic

cells, such as the olfactory bulb or the ventral hypothalamus,

the proportion of ARX/GABA positive cells is very high and

represents approximately 90% of GABAergic neuronal cells

(Fig. 5C). A similar co-expression pattern was observed on

newborn brain sections (data not shown). On the contrary, no

co-expression of Arx and CaMKII was observed on these

sections. These results are in line with those obtained on

primary young and mature cultures of cortical brain neurons.

These data, together with the observed Arx expression pattern

that coincides with known migration routes of interneurons

during embryonic brain development, suggest that Arx is

predominantly expressed in GABAergic neurons and might

be involved in the differentiation or migration or function of

at least a subset of these GABAergic interneurons.

3.6. Phenotypic expression of cells expressing mutated ARX

Different mutations in the ARX gene are involved in

syndromic and non-syndromic forms of mental retardation.

The mutation 98C>T leading to a leucine to proline change

at amino acid 33 of ARX (L33P) is located in the octapep-

tide domain designated as the GEH domain and was

identified in a family with X-linked nonspecific mental

retardation. The mutation 1058C>T is predicted to cause a

change of proline 353 to leucine (P353L) and is one of the

six invariable residues, typical of the paired type homeo-

domain. This mutation has been identified in a rare X-linked

recessive myoclonic epilepsy with spasticity and intellectual

disability. Another mutation is a polyA tract expansion

(insertion of 7 alanines) within the first polyalanine stretch


(at position 100–115) of the ARX protein. This type of

mutation has been identified in a family with X-linked

nonspecific mental retardation with aggressive behaviour.

To examine the cellular consequences of these ARX

mutations, COS7 cells and PC12 cells were transfected with

these three different mutant constructs fused to Green

Fluorescent Protein (GFP) as well as with the wild-type

construct. COS7, as well as PC12 cells do not express the

ARX protein endogenously. ARX wild-type and the three

mutant constructs (ARX-L33P, ARX-P353L and ARX-

polyA) were detected exclusively in the nucleus of the cells

(data not shown). No dramatic changes in PC12 cell

morphology before and after nerve growth factor (NGF)

treatment were noted with any of the mutant fusion proteins.

However, we cannot exclude an effect of the GFP-tag on the

function of the different ARX proteins. Interestingly, the

polyA tract expansion mutation did not lead to abnormal

intranuclear protein aggregation 1, 3 and 7 days after

transfection in COS7 cells or PC12 cells. These observa-

tions were confirmed by absence of insoluble aggregates in

the sensitive SDS-PAGE stacking gel performed on day 1

after transfection (data not shown). Moreover, by counting

the number of transfected cells 1, 3 and 7 days after

transfection with wild-type or one of the three mutant

constructs, we observed that the percentages of living cells

expressing ARX mutants were not significantly different

from those expressing wild-type ARX at any time point

examined. These results suggest that the pathogenic effects

of these mutations cause only subtle changes, if any, in these

cell types.

4. Discussion

We analysed the expression of the Arx protein in the

CNS during development and in the adult. Arx was found in

a number of specific brain regions as well as in the

developing spinal cord. During early development of the

telencephalon, expression of Arx is observed in the ventric-

ular zone and in post-mitotic neurons in the pre-plate. As

development emerges, Arx expression is mainly observed in

the marginal zone (MZ) as well as in post-mitotic neurons

that are migrating through the intermediate zone and the

cortical plate towards their final position. However, as Arx

is also expressed in cells of the ventricular zone, we can

suggest that this protein might play not only a role in post-

mitotic and migrating neurons but also in dividing progen-

itor cells. The Arx positive cells present in the MZ are

different from Reelin expressing Cajal-Retzius cells. It is

known that other types of neurons are present in the MZ, but

thus far their identity has not been fully characterised [15].

A strong Arx expression is present in the mouse devel-

oping striatum and in the intermediate zone of the gangli-

onic eminences, a region where GABAergic interneurons

originate [1,10]. Subsequently, these neurons follow a

tangential migratory route to their positions in the develop-

ing cortex. At least three spatially and temporally distinct

migration routes have been identified [12]. Although it

cannot be excluded that the Arx expression observed in

the pre-plate originates from cells produced in the dorsal

part of the cortex, the observed expression pattern seems

most coherent with a flux of migrating cells derived from

the medial ganglionic eminence that contributes to the ARX

positive interneurons. This suggests that Arx plays a role in

development and/or migration of neuronal cells, likely to be

interneurons derived from the ganglionic eminences. These

observations are reinforced by the expression in young

primary cultures of Arx in GABAergic neurons shown in

this study and the findings reported by Ref. [9]. These

authors revealed that deficiency of Arx in the mouse seems

to inhibit migration of GABAergic interneurons from the

medial ganglionic eminence (MGE) to the intermediate

zone, while the migration of GABAergic interneurons from

the MGE to the cortical subventricular zone though the

lateral ganglionic eminence (LGE) is maintained. In con-

clusion, our expression data obtained in late development

(E13.5 to E16.5) are in total agreement with the observed

findings in Arx � /Y mutant mice [9].

Moreover, these Arx expression data in the developing

mouse cortex and the ganglionic eminences are in line with

the expression pattern described during human brain devel-

opment [17].

During mouse spinal cord development, we observed an

early expression of Arx in the neuroepithelium. At E13.5,

we still found an expression in the floorplate, an interme-

diate target for path-finding commissural axons located at

the ventral midline of the developing mouse spinal cord. In

addition, Arx is expressed in the mantle layer of the

intermediate region (IR) and the prospective dorsal horn.

Interestingly, the IR is defined as the area between the dorsal

and ventral horns that contains primarily interneurons. Other

homeobox-containing transcription factors with restricted

domains of expression in post-mitotic cells of the IR have

been previously identified including En-1, Evx-1, Lmx-1

and Dbx-1 [13]. This observation underlies again the

involvement of Arx in the regulation of yet-to-be identified

neuronal process during CNS development.

In the adult, Arx expression is no longer present in the

spinal cord but noted in all cortical layers, the striatum, the

amygdala, the thalamus, the hypothalamus and olfactory

bulb, both by Western blot analysis and by immunohisto-

chemistry. Interestingly, Arx/CAMKII and Arx/GABA dou-

ble immunostaining experiments on adult rat brain sections

and primary cultures of neuronal and glial cells showed an

expression of Arx in a subset of GABA-expressing neurons,

whereas no expression of Arx in CaMKII excitatory neuro-

nal and glial cells was observed. Although it is difficult to

extrapolate from these results on the putative role of Arx in

adult brain, our results suggest that Arx is probably not

involved in intrinsic function of excitatory-type CAMKII-

positive neurons. Although we cannot exclude a role in

early development of this type of neurons, seen in the high


Arx expression present in the germinal ventricular zone. On

the other hand, Arx expression patterns observed in the

developing brain and the expression of Arx in GABAergic

neurons of adult brain and primary cultures suggest that Arx

is likely to be involved in the differentiation, migration and/

or function of at least a subset of GABAergic interneurons.

However, not all brain areas that contain GABAergic

neurons require Arx expression, as is for example the case

in the cerebellum were Arx expression is absent in the

inhibitory Purkinje cells. In the Dlx1/Dlx2 double mutant,

there is a massive reduction of GABAergic interneurons in

the cerebral cortex [1,2]. This is due to defects in the

different migration routes of the neurons originating from

the ganglionic eminences, resulting in reduced numbers of

striatal (GABAergic and cholinergic), olfactory bulb

(GABAergic and dopaminergic) and cortical (GABAergic)

interneurons [18]. However, as Dlx1/Dlx2 mutants still

express GABA in the subcortical telencephalon [1,2], it is

clear that other genes are involved in the control of

GABAergic neuronal development. Arx is thus likely to

fall into this category.

Our results suggest that the Arx protein is expressed in a

large proportion of young developing neurons, with dimi-

nution of expression in the adult, leaving only some

GABAergic neurons continuing to express this protein.

Moreover, these findings could explain the occurrence of

seizures observed in the great majority of patients with an

ARX mutation, due to a reduced number or function of

cortical GABAergic neurons. GABA, the principal inhibi-

tory neurotransmitter in the cerebral cortex, maintains the

inhibitory tone that counterbalances neuronal excitation.

Abnormalities of GABAergic function have already been

associated with epilepsy in humans. Recently, mutations in

the GABAA-receptor g2 were identified not only in patients

with generalised epilepsies and febrile seizures, but also

with severe myoclonic epilepsy in infancy [3,8]. Mutations

have also been identified in GABRA1 encoding the a1 sub-

unit of the GABAA receptor in affected individuals of a

large family with juvenile myoclonic epilepsy [6]. The high

expression of Arx observed in the amygdala is also concor-

dant with the occurrence of seizures. In fact, alterations in

the density of GABAergic interneurons (especially neurons

which contain calbindin and/or neuropeptide Y) in the

basolateral amygdala have been reported in several models

of epilepsy [14,21].

Different mutations have been identified in the ARX gene

and have been shown to be involved in a broad spectrum of

neurological disorders ranging from non-syndromic to syn-

dromic forms of mental retardation and XLAG [5,9,19].

ARX mutations include missense mutations, polyalanine

tract expansions, nonsense and frameshift mutations. To

better understand the clinical heterogeneity associated with

several missense mutations in ARX including the polyala-

nine expansion, we studied the spatial distribution of GFP-

tagged wild-type and ARX mutants in transfected COS7 and

PC12 cells. All tested mutants were localised in the nucleus

and did not affect the morphology of COS7 and PC12 cells

even after nerve growth factor (NGF) treatment. Moreover,

over-expression of wild-type or mutant ARX did not induce

cell death in vitro and no abnormal protein aggregation was

observed by over-expression of the ARX-polyalanine mu-

tant in COS7 and PC12-cells. This observation is in agree-

ment with previous data concerning alanine stretch

expansions in other disease gene products. In fact among

the alanine-expanded proteins involved in human diseases

(HOXA13, HOXD13, CBFA1, ZIC2, PABPN1), mutated

PABPN1 is the only one that is reported to induce the

formation of intranuclear inclusions [7]. A further analysis

of mutant clones in different cell types is required for a

further understanding of molecular and cellular consequen-

ces resulting from the dysfunction of ARX.

Acknowledgements

The authors are grateful to P. Billuart, J. Parnavelas, F.

Fauchereau, M. Mildalgo-Sanchez and E. Bloch-Gallego for

their helpful comments, M.C. Vinet for her technical

assistance, A. Goffinet for providing the Reelin antibodies

and A. Koulakoff for glial cell cultures. This work was

supported by the European Community (QLG2-CT-1999-

00791, QLG3-CT-2002-01810), CNRS, Fondation pour la

recherche Medicale (FRM), Fondation Bettencourt Schuler,

Fondation France Telecom, and Fondation Franc�aise pour laRecherche sur les Epilepsies K.P. is supported by a PhD

fellowship of the Ministere de la Recherche, H.V.E. is a

post-doctoral researcher of the Fund for Scientific Research-

Flanders, Belgium (FWO-Vlaanderen).

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