6.5 GCG triplet expansion in ARX gene in West syndrome: generation of an animal model suitable for morpho-functional analysis

6.5 GCG triplet expansion in ARX gene in West syndrome: generation of an animal model suitable for morpho-functional analysis

ABSTRACT

ARX (Aristaless-related homeobox gene) is a X-linked gene encoding for a transcription factor containing several well-conserved domains such as a homeobox, an aristaless domain and four polyalanine tracts. ARX expression appears first in Central Nervous System (CNS) starting from early developmental stages, and persists in mammalian cortical GABAergic interneurons up to adulthood thus suggesting an involvement for ARX in the CNS development and function.

It has been reported that mutations of ARX are responsible for the onset of severe pathologies. An increasing number of evidences has linked the expansion of tracts of single aminoacid repeats to human diseases.

Recently, it has been identified an expansion of 7 GCG repeats, ARX(GCG

), resulting in the expansion of 7 alanine residues within the first polyalanine tract in children affected by West syndrome, a severe form of infantile epilepsy characterized by the presence of spasms, hypsarrhythmia and mental retardation. Molecular and cellular mechanisms underlying this pathology are still elusive due to limited availability of pathologic cerebral tissue.

For this reason the aim of this project is the generation of a knock-in allele in which the cDNA of the human ARX gene carrying the expansion of the 7 GCG repeats will replace the murine Arx gene. Recombinant cells thus engineered will be used for the generation of a stable embryonic stem cell line and of a mouse model line, which will be useful tools to improve the understanding of the mechanisms leading to aberrant CNS development and functioning.

6.5.1 INTRODUCTION

Mcerebral cortex develops from the rostral neural tube through a complex

and highly coordinated series of events that follow an inside-out pattern and that lead to the development of a laminated six-layered structure. This process requires the proper cell generation/proliferation, neuronal identity specification, cell migration, and the establishment of appropriate patterns of connectivity between distinct neuronal subtypes. Some of the molecular factors that control the specification of neurons and the patterning of cerebral cortex have been identified (e.g. transcription factors, secreted and cell- surface proteins, growth factors etc.). Mutation in genes involved in each of these tightly regulated processes may prompt to an impairment in the development of cerebral cortex, that in turn may predispose to a variety of clinical consequences, one of which is epilepsy. It has been reported that about 40% of drug-resistant epilepsies are caused by genetic malformation of cerebral cortex, as well as developmental delay and mental retardation.

Epileptic disorders have been grouped into two main cathegories:

epilepsies associated with malformations of cortical development (MCDs) and epilepsies associated with a structurally normal brain (or with minimal abnormalities only) (reviewed in Andrade, 2009). Focal cortical dysplasia, polymicrogyria, heterotopia, lissencephaly and schizencephaly are examples of MCDs. Juvenile myoclonic epilepsy, childhood absence epilepsy, some familial forms of focal epilepsy and epilepsies associated with febrile seizures belong to the latter group.

The importance of Arx (Aristaless-related homeobox gene) in brain development came to light when it was simultaneously identified by two groups of researchers as a key gene responsible for mental retardation and epilepsy (Strømme et al., 2002; Bienvenu et al., 2002).

Arx gene has been isolated in mouse and zebrafish on the basis of its homology with the gene aristaless (al) of Drosophila and subsequently the human orthologous has been identified (Campbell and Tomlinson, 1998).

Arx is localized on the short arm of X chromosome and it is constituted by

five exons. The Arx protein contains several well-conserved domains: a

paired-like homeodomain with a glutamine in position 50, that ensure the

specificity for DNA binding; an aristaless domain (C-peptide), acting as transactivation domain; an octapeptide, with the function of transcriptional repressor and four polyalanine tracts whose function has not been yet fully understood (Mailhos et al., 1998).

In mice, Arx is expressed in somites, testicles and developing CNS (Miura et al., 1997; Ohira et al., 2002). Expression in CNS occurs from early developmental stages in ventral thalamus, dorsal telencephalon and in both lateral and medial ganglionic eminences from which cortical GABAergic interneurons arise (Kitamura et al., 2002; Colombo et al., 2004; Poirier et al., 2004; Yoshihara et al., 2005). Expression of Arx persists in a subpopulation of cortical GABAergic interneurons but not in radially migrating projection cells in the adult brain, thus suggesting a role for Arx in the differentiation of specific neuronal cell types and in the generation and maintenance of functional neuronal networks (Kitamura et al., 2002; Colombo et al., 2004;

Poirier et al., 2004).

Kitamura and collaborators have demonstrated that Arx is involved in cortical cell proliferation (Kitamura et al., 2002). They have produced a mouse knock-out replacing Arx exon 2 with LacZ and observed a phenotype very similar to the human XLAG. Hemizygote died within half a day of birth, and were characterized by a smaller brain and smaller olfactory bulbs.

Pulse-chase experiments with bromodeoxyuridine revealed decreased proliferation in the VZ of the cortex, most probably contributing to the reduction in its size. Moreover, double-labelling experiments have revealed that a population of cells in the forebrain co-expressed Arx and neuropeptide Y. These cells were absent from the striatum, neocortex and hippocampus of mutant mice, suggesting that Arx may play a role in the differentiation of these neurons (Kitamura et al., 2002; Yoshihara et al., 2005). However, due to early lethality of mutant mice information about the role of Arx in the mature GABAergic interneurons are still missing.

Molecular and clinical studies in humans have revealed that mutations

in ARX gene are responsible for severe pathologies either with evident

malformations or without. Missense or nonsense mutations in ARX homeodomain originate pathologies belonging to the former group such as XLAG (X-linked lissencephaly with abnormal genitalia) (Kitamura et al., 2002; Bonneau et al., 2002) and Proud (Kato et al., 2004) syndromes, while, in the latter group, pathologies such as Partington or West syndromes are caused by mutations outside the homeodomain region or by expansion of polyalanine tracts (Strømme et al., 2002). Indeed, there is growing experimental support for the involvement of polyalanine expansions in the onset of pathologies associated with mental retardation and CNS malformations (Albrecht and Mundlos, 2005). To date, nine genes with alanine tract expansions have been described, most of them coding for transcription factors that play important roles during development.

Polyalanine expansion-derived conditons have many features in common, suggesting shared pathogenetic mechanisms. In particular, recent studies have grouped polyanlanine expansion pathologies into the category of diseases caused by protein misfolding (Albrecht and Mundlos, 2005).

Proteins that do not fold correctly are usually recognized by chaperones and rapidly degradated, but in some cases misfolded protein can elude this tight control and accumulate. The main problem is that not-yet folded proteins are sticky, because their hydrophobic amino acids are not yet buried completely.

As a result, they may clump together forming cellular aggregates causing profound cellular dysfunctions and, thus, disease.

West syndrome may be caused by an expansion of 7 GCG repeats

(GCG)

, coding for alanine residues, into the second exon of the ARX

gene, within the first polyalanine tract (Strømme et al., 2002; Wohlrab et al.,

2005). West syndrome affect babies under one year of age and consists of

extension and flessure spasms infantile spasms associated with ocular

deviation, an electroencephalogram pattern of hypsarrhythmia, severe

epilepsy and subsequent mental retardation. Molecular and cellular

mechanisms underlying this pathology are still elusive due to limited

availability of pathologic cerebral tissue from autoptic matherial.

In the last few years results obtained from in vitro studies have suggested that the mutated form of ARX, ARX(GCG)

, promotes the formation of intranuclear inclusions which in turn cause an increase in cell death (Nasrallah et al., 2004; Shoubridge et al., 2007). Apart from these results derived from in vitro studies, the molecular and cellular mechanisms underlying West syndrome are to date ignored.

6.5.2 MATERIALS AND METHODS ES cell culture

I used the feeder-independent ES cell line E14Tg (BayGenomics), derived from the 129/Ola strain of mice (Nichols et al., 1990). The ES cell line was maintained on gelatinized tissue culture dishes in ES cell medium containing the leukaemia inhibitory factor (LIF) according to BayGenomics protocols available online <http://baygenomics.ucsf.edu/>.

Electroporation of ES cells

The ARX(GCG)

targeting vector was purified on a Qiagen column kit and was digested with NdeI. The linearized DNA was purified with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and dissolved in sterile water at a concentration of 1 mg/ml.

The day of the electroporation, the cells were 80% confluents and the

medium was changed 2 hours before the cells are harvested. The ES cells

were trypsinized, resuspended at a concentration of 2 x 10

cells/ 0.7 ml of

PBS and electroporated with 30 µg of linearized targeting vector, by using a

Bio-Rad Gene Pulser unit using the Time Constant Protocol at a setting of

0.2 msec and 800 volts. After pulsing, the results screen indicated a

capacitance of 10 microF and infinite resistance. Electroporated ES cells

were recovered in the cuvette for 20 minutes at RT and then transferred in

40 ml of ES cell medium. 10 ml of ES cell medium with about 5 × 10

cells

were plated into 10 cm diameter tissue-culture petri dishes. The following

day the medium was replaced with medium containing 12.5 g/ml active

geneticin (G418). The medium was changed daily. About 7 days after electroporation cells that have integrated the targeting vector form colonies about 1 mm in size and were picked. Recombinant resistant colonies were picked and replicated in 96-well plates. Clones contained in one replica-plate were stocked at -80°C while clones contained in the second set of plates were used for Southern blot analysis.

About 7 days after electroporation recombinant resistant cells have formed colonies that were isolated by picking. Clones were screened by PCR using the following oligonucleutides

Recombinant (forward) 5’-

-3’

Recombinant (reverse) 5’-

-3’

Wild-type (forward) 5’-

-3’

Wild-type (reverse) 5’-

-3’

Southern blot assay

Once picked, the ES recombinant clones were expanded according to BayGenomics online protocols. Total genomic DNA was extracted from each of the ES cell clones with phenol-chloroform and ethanol precipitation, digested with specific restriction enzymes and separated according to size by gel-electrophoresis, lifted to a nylon filter and hybridized with a proper radiolabeled probe.

In particular, to screen for ARX(GCG)

targeting vector homologous recombination, ES cells DNA was digested with EcoRI and a probe external to the left homology arm was used to screen for the 5’ correct recombination.

The size of the wild-type band was 10.6 Kb while the recombinant band size

was 5.8 Kb. ES cells DNA digested with KpnI, instead, was analyzed with a

probe external to the right homology arm to screen for the 3’ correct

recombination. The size of the wild type band was 7.3 Kb while the

recombinant band size was 5.0 Kb. A neo internal probe was also used to

exclude the possibility of additional integrations (not shown).

Karyotype analysis

1-2 x10

ES cells are seeded in a 60 mm gelatinized plate and culture o/n.

Then, 0.06 µg/ml of colcemid is added to culture medium for 2-3 hours and is washed with 2 ml of PBS. Cells are collected in a 15ml tube and are then incubated in 1 ml of trypsin/EDTA for 5 minutes, which is inactivated adding 4 ml of medium. Cells are resuspended carefully by pipeting up and down to obtain a single cell suspension and then pelleted at 1000 rpm for 5 minutes and resuspended again. 5 ml of hypotonic solution (0.56% KCl in water) is added and cells are incubated 6 minutes a RT to swell the cells. Cells are pelleted, the hypotonic solution is removed and cells are resuspended very carefully. 5 ml of cold fixative solution (3 methanol:1 glacial acetic acid) is added pipetting several times to avoid formation of clumps and incubated 20 minutes a RT. Cells are pelleted again, resuspended in 5 ml of fresh fixative and incubated 5 minutes a RT twice. Then slides are preparated: slides are washed in acetone and then in water and air-dried. After dipping in ice-cold 40% methanol slides are pulled out and the excess of liquid is drain off without shaking. Hold the slides at 45° angle 1-3 drops of cell suspension are dropped from about 40-50 cm height with a 1 ml pipetman. Then slides are dried o/n and the next day are placed 5 minutes in 5N HCl, rinsed for 5 minutes by constant flow of distilled H

O, then stained with Giemsa dye for 40 minutes. The chromosome can be also visualized with DAPI staining.

Chromosomes were then manually counted to exclude aneuplidy.

ES cell blastocyst injections and mouse breeding

ES cell lines were thawed and passed for 6 days in ES cell medium in the

absence of G418. The day of injection, the medium was changed several

hours before harvesting the cells. A confluent 25 cm

flask was trypsinized

for 3-4 minutes and diluted into 9 ml of cold ES cell medium without LIF,

pelleted and resuspended in 0.8 ml of ES cell medium (without LIF) in a

sterile 1.5 ml screw-top microcentrifuge tube. Before they were added to the

injection chamber, cells were kept on ice (for up to several hours) to prevent clumping.

Blastocysts were flushed from pregnant C57BL/6 females and collected into a CO

-independent medium containing 10% FBS. Blastocysts were expanded for 1-2 hours in ES cell medium in a 37°C / 6% CO

incubator, transferred to a hanging drop chamber, and cooled to 4°C. ES cells were added to the hanging drops and the blastocysts were injected with enough cells (20 or more) to fill the blastocoele. Injected blastocysts were then transferred to pseudopregnant recipient females (10-15 blastocysts/uterine horn). Typically, injection of 10 blastocysts will yield an average of twice male chimeras with germline mosaicism. Of note, the 129/Ola cells carry the recessive pinkeye (p) and chinchilla (cch) mutations;

strong chimeras exhibit patches of cream coloured fur and can have pink eyes.

Vasectomy

Vasectomized males are required to generate pseudopregnant recipients females.

Vasectomy is performed on CD1 males mice at 6-8 weeks of age. 10- 14 days after vasectomy, the animals were tested to verify their sterility. One or two fertile female mice were placed with the vasectomized male and were checked for plugs the following morning. Females with plug were sacrificed and their oviducts were flushed 24 hours later. The eggs should be at one cell stage or unfertilized because whether they are at two cells stage, the vasectomy is not performed correctly.

Genotyping

A sample of tissue from the tail tip of each animal was obtained by tail

biopsy. The tissue was used for the extraction of the genomic DNA. In order

to identifiy animals carrying mutation a PCR analysis using specific primers

is performed.

ES cells culturing

Undifferentiated ES cells were grown on gelatin-coated tissue culture plates in ES cell medium in the presence LIF according to BayGenomics protocols available online <http://baygenomics.ucsf.edu/>.

One step protocol in defined medium (Fico et al., 2008)

ES cells were maintained in an undifferentiated state culture and in vitro. At day 0, ES cells were dissociated in a single-cell suspension and 1000 cells/cm

were plated on gelatin-coated plates (diameter from 10 to 0,8 cm).

The culture medium was replaced daily during differentiation process.

Culture Media composition ES cell medium

G-MEM

FCS (Fetal Calf Serum) 10%

L-Glutamine 2 mM

Penicillin-Streptomycin 100 U/ml 2-Mercaptoethanol 0.05 µM Non-essential aminoacids 1 mM Sodium pyruvate 1 mM

LIF (Leukemia Inhibitory Factor) 1000 U/ml

KSR medium

KnockOut DMEM (Invitrogen)

Knockout Serum Replacement (KSR, Invitrogen) 15%

L- Glutamine 2 mM

Penicillin-Streptomycin 100 U/ml 2-Mercaptoethanol 0.1 mM

Immunocytochemistry

Cells were fixed in directly on the plate in 4% in PBS for 15 minutes at RT, rinsed twice in PBS and processed for immunostaining. For immunostaining, sections were incubated with primary antibodies in PBS containing 5% heat- inactivated lamb serum and 0.1% Triton X-100, o/n at 4°C. Samples were washed with PBS and 0.1% Triton X-100 all day and then incubated with secondary antibodies in PBS containing 5% heat-inactivated lamb serum and 0.1% Triton X-100, o/n at 4°C.

Antibodies and dilutions were as follows: rabbit anti-GABA (Sigma) 1:1000; Rhodamine Red-X goat anti-rabbit IgG (Invitrogen) 1:500

6.5.3 RESULTS AND DISCUSSION

Generation of the ARX(GCG)

allele by homologous recombination in ES cells

The targeting vector hARX(GCG)

was generated starting from the right homology arm (RA) (Fig. 1): by means of PCR strategy, a fragment corresponding to part of the exon 2 and part of the intron 2 of the Arx mouse gene was amplified, resulting in a 2.5 Kb fragment (Fig. 1). Subsequently, a PGKneo cassette, which contains the geneticin resistance gene flanked by two loxP sites, was inserted within the SacII site upstream the RA. A plasmid containing the myc-TAG and the cDNA of human ARX carrying the expansion of the 7 GCG repeats (pCMVmyc/hARX(GCG)

) was a kind gift of Dr. Gécz. The construct was verified by sequencing and then a ApaI/HpaI restriction fragment, comprising both the myc-TAG and the hARX(GCG)

cDNA, was cloned upstream the PGKneo cassette. The left homology arm (LA) was obtained using the same procedure as the RA, by means of PCR.

The sequence corresponds to a 2.9 region at the 5’ end of the mouse Arx

ATG translation starting site and was cloned within the ApaI restriction site

upstream the myc-TAG, generating the ARX(GCG)

plasmid. In this way,

the homologous recombination event can remove exon 1 and part of exon 2

of the endogenous Arx mouse gene, ensuring the lack of its expression.

Each step of the targeting vector preparation was analysed by restriction pattern analysis and DNA sequencing.

Figure 1 Generation of the ARX(GCG)10+7 allele. A schematic representation of the Arx locus (a), the targeting vector (b) and the recombinant allele (c) are shown.

The targeting vector contains the hARX(GCG)10+7 cDNA (yellow) with a myc-TAG (red) upstream for the localization of the human mutated protein. A PGK-neo cassette (pink), flanked by two loxP sites, was cloned downstream the gene in order to select recombinant ES cell clones. Two homology arms are present at both of the terminal ends of the construct, in order to favor the homologous recombination event within the murine Arx locus. (b) Southern Blot analysis confirms homologous recombination in ES cell clone 294 (EcoRI and KpnI digests), and (c) kariotype analysis shows a correct chromosome asset of ARX(GCG)10+7 ES cells.

a

b c

The following step was the introduction via electroporation of the NdeI linearized targeting vector in mouse Embryonic Stem (ES) cells. The feeder- independent ES cell line E14Tg, derived from the 129/Ola strain of mice was used (Nichols et al., 1990). Cells were maintained on gelatinized tissue culture dishes in ES cell medium containing leukemia inhibitory factor (LIF), according to BayGenomics protocols available online

<http://baygenomics.ucsf.edu/>.

After one week of geneticin (G418) selection, recombinant clones expressing the neomycin resistance gene formed colonies, which were picked and replicated in 96-well plates. Clones contained in one replica-plate were stocked at -80°C while clones contained in the second set of plates were amplified and used for Southern Blot analysis to screen for proper 5’

and 3’ recombination within the Arx locus. I performed the first screening by means of Southern Blot analysis using a probe against the left homology arm to verify the correct recombination at the 5’ end: after several rounds of electroporation I could isolate and screen a total number of 3072 G418- resistant clones. Finally, among the 960 clones screened in the last round of electroporation, one ES cell clone (clone n. 294) showed the expected 5.8 Kb band (Fig.). Screening clone 294 for recombination at the 3’ end showed the expected 5.0 Kb band, confirming, on the whole, that genomic DNA of ES cell clone n.294 was engineered so that the human ARX cDNA, harbouring the expansion [ARX(GCG)

] found in West syndrome patients, replaces the endogenous Arx gene.

ARX(GCG)

ES cells karyotype analysis

It is known that long-term culture and in vitro manipulation of ES cells can affect chromosomes number and integrity and can cause loss of totipotency.

As a consequence, these ES cells can fail in obtaining contribution to all

tissues of chimeric mice, including the germ line. To assess the absence of

chromosomal abnormalities in recombinant ES cells I performed a karyotype

201 analysis of metaphase chromosomes. The recombinant ES clone n. 294 showed a normal chromosomal asset, with 19 autosomal pairs and 2 sex chromosomes. However, chromosome counting does not account for chromosomal aberrations or spontaneous mutations, and clones with a normal chromosomal asset may still not contribute to germline transmission.

Fig.2: Blastocyst microinjection and generation of ARX(GCG)10+7 mouse chimeras. (a-c) Microinjection main steps: blastocysts are captured by the holding pipette and then the injection pipette is used to collect some ARX(GCG)10+7 ES cells (a). Next, the injection pipette is used to puncture the blastocyst and the ES cells were expelled into the blastocoel cavity (b,c). After injection, blastocysts are re-implantated in pseudopregnant females in order to obtain chimeras (schematic shown in d). Chimeric pups thus generated show the typical patchy coat-colour with cream colour fur derived from the recombinant ES cells and the brown colour fur derived from the host blastocyst (e).

ARX(GCG)

ES cells microinjection and generation of chimeric mice The following step has been the microinjection of the ARX(GCG)

knockin ES cells into host blastocysts in order to generate mouse chimeras. It is important to remember that the cells of the host embryo differ from the ES cells used for the generation of the ARX(GCG)

line in terms of genetic background: the former are derived from the SV129/Ola strain characterized

♂

ARX(GCG)

chimera

♀ wild-type

(wt)

♂,

♀ ♀ ♂

X

X 


a b c

d e

by a chinchilla coat-colour, while the latter are derived from the black-coated C57BL/6 strain (Fig. 2). E3.5 blastocysts were obtained from pregnant C57BL/6 females and were injected with an average of 20 ES cells per blastocyst. The chimeric blastocysts thus obtained were transferred into the uterus of E2.5 CD1 pseudopregnant females. In average, 12 injected blastocysts were transferred in each foster mother. To date, three different rounds of microinjection have been performed. The first one resulted in a total of 218 chimeric blastocysts reimplanted into 18 females, which gave birth to 28 pups. 9 out of the 28 pups showed the typical chimeric patchy coat-colour with cream colour fur derived from the recombinant ES cells and the brown colour fur derived from the host blastocyst, meaning that ARX(GCG)

knockin ES cells injected contributed to various tissue and organs of the chimeras. During the second round we microinjected 72 selected blastocysts that gave rise to additional 7 chimeric mice out of a total number of 12 pups born. The third round of microinjection resulted in a total number of 110 blastocysts injected. Among the 19 pups born, 5 resulted to be chimeric mice. PCR analysis confirmed the presence of the recombinant allele in the 21 chimeric animals (18 males, 3 females) obtained.

Table1

Assessment of germ line transmission in ARX(GCG)

mouse

chimeras

Chimeric mice thus generated were intensively mated with wild-type mice in order to screen them for germ line transmission. In particular, crossing ARX(GCG)

mouse chimeras should allow to obtain females heterozygous for the ARX(GCG)

expanded form. These females, as in humans, are expected to show a normal phenotype, so that crossbreeding heterozygous females to wild-type males will allow to obtain hemizygous males, harbouring the ARX(GCG)

allele. Hemizygous mouse males will likely present those phenotypic alterations observed in West syndrome patients.

Results are schematically recapitulated in Table 1. To date, among 734 pups generated from mouse chimeras, only 6 pups (4 males and 2 females), all obtained from a single chimeric mouse showed the expected agouti fur. Even though unfortunately none of them harboured the recombinant allele, obtaining these mice demonstrates that chimeras germline is contributed by SV129/Ola-derived ARX(GCG)

ES cells.

Unfortunately, obtaining agouti wild-type females means that ARX(GCG)

ES clone n. 294 was constituded also by SV129/Ola wild-type cells,

The disappointing outcome of our effort in the generation of the myc-

tag-ARX(GCG)10+7 mouse line may have more than one possible

explanation. One reason for the lack of germ line transmission could reside

intrinsically in the nature of the sole myc-tag-ARX(GCG)

knockin ES

clone we identified, whose potency to generate germ cells might have been

strongly reduced, even though the presence of a correct karyotype was

confirmed. Myc-tag-ARX(GCG)

ES cells produced chimeras with a

variable percentage of coat color chimerism, but crossing chimeric mice with

C57BL/6 animals, no heterozygous agouti offsprings were produced. Litter

size and composition were normal suggesting that mutant embryos were not

being produced, rather than that they were dying during embryonic

development. However, as ARX gene is involved in early events of

embryogenesis, we wanted to exclude that the presence of the myc-tag-

myc-tag-ARX(GCG)

allele could induce early developmental lethality both

a

in hemizygous males and heterozygous females. For this reason, I screened using PCR genotyping assay numerous embryos at 9.5 day of gestation and, similarly to what observed in newborns, I did not find any positive for the myc-tag-ARX(GCG)

allele. Another possible explanation could be that mutant-bearing spermatids or later gametes, if generated from the chimera, might not be surviving due to Arx expansion. This would be consistent with the reported expression of Arx in developing testis.

In vitro differentation of ARX(GCG)

cells

Taken into account the difficulty to generate the ARX(GCG)

mouse line, I decided to address the question about the molecular consequences due to ARX(GCG)

expansion performing in vitro differentiation experiments, driving ARX(GCG)

ES cells toward GABAergic internerneuron phenotype. For this purpose, I used a single-step protocol to differentiate ES cells in neurons developed by Fico and collegues (Fico et al., 2008). Using this protocol, which is based on a defined medium containing 15% knock-out serum replacement, I could efficiently obtain GABAergic interneurons, within a mixed cell population, both from wild-type and ARX(GCG)

ES cells.

Preliminary data on GABA-positive neurons show that GABAergic interneurons obtained in vitro from ARX(GCG)

ES cells seem to have arborisations with higher complexity than those obtained from wt cells (Fig.

3). Wild-type GABAergic neurons grown in low-density areas showed usually a bipolar-like structure, with two main neurites extending from the cell soma.

Conversely, ARX(GCG)

derived GABAergic neurons showed at least

three neuritis, that in some cases could further branch (Fig. 3). In some

areas, differentiating cells form colony-like aggregates, which are connected

one to the other by long fascicles of fibers. The tendency of ARX(GCG)

cells to develop more axons is maintained also in this case, as they showed

more fascicles bridging adjacent colonies, as compared to wild-type derived

neurons (Fig. 3).

Figure 3: In vitro differentiation of ARX(GCG)10+7 ES cells toward GABAergic interneurons. (a) Schematic representation of ES cell differentiation towards neurons with a low confluence protocol. GABA-positive neurons were efficiently obtained from wild-type and ARX(GCG)10+7 ES cells after 14 days culture in KSR supplemented medium. (b-d) GABA immunostaining on GABAergic interneurons obtained from wild- type (b,d) or ARX(GCG)10+7 ES cells (c,e). Preliminary data on ES cell differentiation show that ARX(GCG)10+7-derived GABAergic interneurons display more neuritis as compared to wild-type-derived GABAergic neurons (b,c). Moreover, ARX(GCG)10+7- derived GABAergic interneurons showed more fascicles bridging adjacent colonies, as compared to wild-type derived neurons (d,e).

For this reason, a more detailed analysis measuring specific

quantitative parameters including number of branches, number of branching

points and branch length will shed light on the morphological phenotype of

ARX(GCG)

ES cells.

It is known that different subtypes of GABAergic interneurons express different genetic marker, such as parvalbumin, calretinin or calbindin (Freund and Buzsaki, 1996). While parvalbumin interneurons tend to be larger and have elaborate axons, calretinin interneurons are smaller and bipolar (Wonders and Anderson, 2006). It will thus be interesting to see whether GABAergic interneurons differentiated in vitro from ARX(GCG)

cells show a peculiar array of genetic markers, as compared to wild-type derived cells.

In parallel, in order to address the effects of polyalanine expansion

(GCG)

in ARX gene, we used an in utero electroporation approach in

collaboration with Prof. Represa’s group at the Institut de Neurobiologie de la

Méditerranée (INMED) in Marseille. This study provided the evidence that

epilepsy in mice expressing ARX(GCG)

would result from a glutamate

network remodeling rather than from alteration in GABA-neuron migration

and maturation. These results are the subject of a manuscript, currently

under revision at Cerebral Cortex, that is enclosed in chapter 5.6.

6.5.4 REFERENCES

Albrecht, A., Mundlos, S. (2005). The other trinucleotide repeat:

polyalanine expansion disorders. Curr Opin Genet Dev.15, 285-293.

Andrade, D. M. (2009). Genetic basis in epilepsies caused by malformations of cortical development and in those with structurally normal brain. Hum Genet 126, 173-93.

Bienvenu, T., Poirier, K., Friocourt, G., Bahi, N., Beaumont, D., Fauchereau, F., Ben Jeema, L., Zemni, R., Vinet, M.C., Francis, F., Couvert, P., Gomot, M., Moraine, C., van Bokhoven, H., Kalscheuer, V., Frints, S., Gecz, J., Ohzaki, K., Chaabouni, H., Fryns, J.P., Desportes, V., Beldjord, C., Chelly, J . (2002) ARX, a novel Prd-class- homeobox gene highly expressed in the telencephalon, is mutated in X- linked mental retardation. Hum Mol Genet. 11, 981-991.

Bonneau, D., Toutain, A., Laquerriere, A., Marret, S., Saugier-Veber, P., Barthez, M.A., Radi, S., Biran-Mucignat, V., Rodriguez, D., Gélot, A.

(2002) X-linked lissencephaly with absent corpus callosum and ambigous genitalia (XLAG): clinical, magnetic resonance imaging, and neuropathological findings. Ann. Neurol., 51, 340 - 349.

Bruneau, S., Johnson, K. R., Yamamoto, M., Kuroiwa, A., Duboule, D.

(2001) The mouse Hoxd13spdh mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes. Dev. Biol. 237, 345-353.

Campbell, G., Tomlinson, A. (1998). The roles of the homeobox genes aristaless and Distal-less in patterning the legs and wings of Drosophila.

Development 125, 4483-4493.

Colombo, E., Galli, R., Cossu, G., Gécz, J., Broccoli, V. (2004) Mouse orthologue of ARX, a gene mutated in several X-linked forms of mental retardation and epilepsy, is a marker of adult neural stem cells and forebrain GABAergic neurons. Dev. Dyn., 231, 631 - 639.

Fico, A., Manganelli, G., Simeone, M., Guido, S., Minchiotti, G., Filosa,

S. (2008). High-throughput screening-compatible single-step protocol to

differentiate embryonic stem cells in neurons. Stem Cells Dev. 17, 573- 584.

Freund, T.F., Buzsàki, G. (1996). Interneurons of the hippocampus.

Hippocampus 6, 347-470

Hino, H., Araki1, K., Uyama E., Takeya M., Araki, M., Yoshinobu K., Miike1, K., Kawazoe1, Y., Maeda, Y., Uchino M., Yamamura K. (2004) Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy. Hum. Mol. Genet. 13, 181-190.

Kato, M., Das, S., Petras, K., Kitamura, K., Morohashi, K., Abuelo, D.N., Barr, M., Bonneau, D., Brady, A.F., Carpenter, N.J., Cipero, K.L., Frisone, F., Fukuda, T., Guerrini, R., Iida, E., Itoh, M., Feldman Lewanda, A., Nanba, Y., Oka, A., Proud, V.K., Saugier-Veber, P., Schelley, S.L., Selicorni, A., Shaner, R., Silengo, M., Stewart, F., Sugiyama, N., Toyama, J., Toutain, A., Lia Vargas, A., Yanazawa, M., Zackai, E.H., Dobyns, W.B. (2004) Mutations of ARX are associated with striking pleiotropy and consistent genotype - phenotype correlation. Hum.

Mutat., 23, 147 - 159.

Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A., Kusaka, M., Omichi, K., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K., Matsuo, M., Kamijo, S.I., Kasahara, M., Yoshioka, H., Ogata, T., Fukuda, T., Kondo, I., Kato, M., Dobyns, W.B., Yokoyama, M., Morohashi, K.I. (2002) Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans. Nat. Genet., 32, 359 – 369.

Mailhos, C., André, S., Mollereau, B., Goriely, A., Hemmati-Brivanlou, A., Desplan, C. (1998). Drosophila Goosecoid requires a conserved heptapeptide for repression of paired-class homeoprotein activators.

Development 125, 937-947

Migliarini, S., Pacini, G., Pasqualetti M. (2008). Generation of a

Tph2/eGFP knockin mouse line for the study of the role of serotonin

during the central nervous system development. Serotonin Club Meeting , Oxford, july 17-20.

Miura, H., Yanazawa, M., Kato, K., Kitamura, K. (1997). Expression of a novel aristaless related homeobox gene ‘Arx’ in the vertebrate telencephalon, diencephalon and floor plate. Mech Dev., 65, 99 - 109.

Nasrallah, I. M., Minarcik, J. C., Golden, J. A. (2004) A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death. J. Cell Biol., 167, 411 - 416.

Ogata, T., Matsuo, N., Hiraoka, N., Hata, J. (2000) X-linked lissencephaly with ambiguous genitalia: delineation of further case. Am. J. Med. Genet.

94, 174 - 176.

Ohira, R., Zhang, Y. H., Guo, W., Dipple, K., Shih, S. L., Doerr, J., Huang, B. L., Fu, L. J., Abu-Khalil, A., Geschwind, D., McCabe, E. R. B. (2002) Human ARX gene: genomic characterization and expression. Mol. Genet.

Metab., 77, 179 - 188.

Pasqualetti, M., Ren, S. Y., Poulet, M., LeMeur, M., Dierich, A. and Rijli F. M. (2002). A Hoxa2 knockin allele that expresses EGFP upon conditional Cremediated recombination. Genesis 32, 109-111.

Poirier, K., Van Esch, H., Friocourt, G., Saillour, Y., Bahi, N., Backer, S., Souil, E., Castelnau-Ptakhine, L., Beldjord, C., Francis, F., Bienvenu, T., Chelly, J. (2004) Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons. Brain Res. Mol. Brain Res., 122, 35 - 46.

Shoubridge, C., Cloosterman, D., Parkinson-Lawerence, E., Brooks, D., Gécz, J. (2007) Molecular pathology of expanded polyalanine tract mutations in the Aristaless-related homeobox gene. Genomics, 90, 59-71.

Stromme, P., Mangelsdorf, M. E., Shaw, M. A., Lower, K. M., Lewis, S.

M., Bruyere, H., Lutcherath, V., Gedeon, A. K., Wallace, R. H.,

Scheffer, I. E., Turner, G., Partington, M., Frints, S. G., Fryns, J. P.,

Sutherland, G. R., Mulley, J. C., Gécz, J. (2002) Mutations in the human

ortholog of Aristaless cause X-linked mental retardation and epilepsy.

Nat. Genet., 30, 441 -445.

Wohlrab, G., Uyanik, G., Gross, C., Hehr, U., Winkler, J., Schmitt, B., Boltshauser, E. (2005) Familial West syndrome and dystonia caused by an Aristaless related homeobox gene mutation. Eur. J. Pediatr. 164, 326- 328.

Wonders, C.P., Anderson, S.A. (2006) The origin and specification of cortical interneurons. Nat Rev Neurosci. 7(9), 687-696.

Yoshihara, S., Omichi, K., Yanazawa, M., Kitamura, K., Yoshihara, Y.

(2005) Arx homeobox gene is essential for development of mouse

olfactory system. Development 132, 751 - 762.