hum. mol. genet. 2013 lee hmg ddt503

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2

Abstract

Intellectual disability (ID) is a highly prevalent disorder that affects 1-3% of the

population. The Aristaless-related homeobox gene ( ARX ) is a frequently mutated X-

linked ID gene and encodes a transcription factor indispensable for proper forebrain,

testis and pancreas development. Polyalanine expansions account for over half of all

mutations in ARX and clinically give rise to a spectrum of ID and seizures. To

understand how the polyalanine expansions cause the clinical phenotype we studied

mouse models of the two most frequent polyalanine expansion mutations ( Arx(GCG)7

and Arx432-455dup24

). Neither model showed evidence of protein aggregates, however a

marked reduction of Arx protein abundance within the developing forebrain was

striking. Examining the expression of known Arx target genes, we found a more

prominent loss of Lmo1 repression in Arx(GCG7)/Y

compared to Arx432-455dup24/Y

mice at

12.5 dpc and 14.5 dpc, stages of peak neural proliferation and neurogenesis,

respectively. Once neurogenesis concludes both mutant mouse models showed similar

loss of Lmo1 repression. We propose that this temporal difference in the loss of Lmo1

repression may be one of the causes accounting for the phenotypic differences

identified between the Arx(GCG)7

and Arx432-455dup24

mouse models. It is yet to be

determined what effect these mutations have on ARX protein in affected males in the

human setting.



3

Introduction

Aristaless-related homeobox gene ( ARX ) [NM_139058.2] (MIM 300382) is a member

of the paired-type homeodomain transcription factor family with critical roles in

development. ARX is an important disease-causing gene on the X-chromosome

contributing to intellectual disability and epilepsy in males (1). Intellectual disability

is a complex debilitating condition of considerable medical importance, with as many

as 1 in every 50 people affected world-wide (2). When the causative gene is on the X-

chromosome, it is referred to as X-linked intellectual disability (XLID). Mutations in

ARX, one of over 100 XLID genes (1) leads to a broad spectrum of ID and epilepsy

phenotypes (3).

ARX is one of eight transcription factors in which expansions of polyalanine tracts

cause hereditary diseases, many with neurocognitive phenotypes (4). Over half

(63/114, 55%) of all reported ARX mutations lead to expansion of the first and

second polyalanine tracts. The predicted mechanism of protein dysfunction for the

autosomal dominant disorders due to expanded polyalanine tracts ranges from gain of

function, complete or partial loss of function and even a dominant negative effect

contributing to associated disease features (5). In contrast, both ARX and SOX3 are

located on the X-chromosome and are subject to X-inactivation in females, making it

difficult to ascertain if the expanded polyalanine tract mutations may be causing

disease due to a dominant gain of function or due to altered or loss of function. In the

case of ARX , a complete loss of function is unlikely given the mutations leading to

complete loss of ARX function result in severe brain malformation phenotypes,

including lissencephaly, hydranencephaly and agensis of the corpus callosum (3, 6,

7).



4

There is a common belief based on studies of some proteins with polyalanine

expansions that these tracts, above a certain threshold, may induce mis-folding and

aberrant protein interactions, degradation, mis-localisation and likely aggregation

under light microscopy (4, 8-11). The polyalanine thresholds at which these events

occur vary between proteins, but are a common finding in over-expression studies in

routine and explant cell culture (4, 8, 9, 12-14). However, the existence and the

contribution of these aggregates to the pathogenesis of disease and ARX-related ID

and epilepsy in particular, remains to be demonstrated. Particularly, there is no data

on in-vivo aggregation of ARX protein available.

Currently, there is one mouse model for the most common ARX mutation, a

polyalanine expansion (from 12 to 20 Ala) in the second tract (6). Moreover, two

independent mouse models have been generated for the expansion (from 16 to 23 Ala)

in the first polyalanine tract, the second most common ARX mutation (6, 15). Patients

with these mutations present with a highly variable phenotype, ranging from mild ID

as the only feature to ID with early onset of epileptic seizures (Ohtahara Syndrome).

Accordingly, these mouse models recapitulate many of the phenotypic presentations

of human patients (6, 15). However, at the molecular and cellular level, it is still

unclear whether protein aggregation or protein mis-localisation are the underlying

drivers. Kitamura et al. (2009) reported only nuclear localisation of Arx in 12.5 days

post-coitum (dpc) ganglionic eminence (GE)-originated migrating cells and in

18.5dpc cortical interneurons in the two mouse models, Arx(GCG)7

and Arx432-455dup24

,

that recapitulate the respective polyalanine tract 1 (c.304ins(GCG)7) and tract 2

(c.432_455dup) mutations (6). In contrast, Price et al. (2009) reported an increased



5

Arx cytoplasmic localisation in the cortical neurons from adult brains of their

independently generated mouse model of the c.304ins(GCG)7 mutation. Neither study

found support for expanded Arx polyalanine aggregate formation.

Patients with the ARX c.304ins(GCG)7 mutation present with severe ID and epilepsy

compared to a milder phenotype in most patients with the c.423_455dup mutation (3).

These differences are at least to some extent recapitulated in the mouse models of the

two mutations with the c.304ins(GCG)7 model having elevated seizure susceptibility

and profound learning impairment (6, 15). Loss of interneuron subsets was shown in

both Arx(GCG)7 mouse lines (6, 15). However, the underlying molecular mechanism for

this phenotypic variation remains to be determined.

We have performed comparative analysis of the brain development of Arx(GCG)7

and

Arx432-455dup24

mouse models. We found no evidence of apparent Arx protein

aggregation or protein mis-localisation under light microscopy. However, we found a

significant reduction of protein abundance for both Arx mutant proteins. Moreover,

we demonstrated a more prominent loss of Lmo1 repression, a well-characterised Arx

direct target, in Arx(GCG)7/Y

compared to Arx432-455dup24/Y

mice. We propose the reduced

Arx protein leading to aberrant expression of Lmo1 as a potential molecular cause of

the ARX polyalanine expansion mutations pathology and variability of clinical

expressivity.



6

Results

No evidence of protein aggregation in-vivo

We performed comparative analysis of Arx(GCG)7

and Arx432-455dup24

mouse models (6)

by studying 12.5dpc Arx protein expression between the wildtype (WT) and the two

mutant mouse models at the dorsal telencephalon (presumptive cortex), ventral

thalamus (VTh), lateral ganglionic eminence (LGE) and the medial ganglionic

eminence (MGE) by immunofluorescence (Figure 1A and E). Arx protein was

detected in cells residing at the mantle zone (MZ) but not the proliferative ventricular

zone (VZ) of the LGE and MGE (Figure 1A and E). Similar strong MZ Arx

expression was also observed in the VTh and HTh. In contrast, a lower but uniform

level of Arx was present within the VZ of the presumptive cortex and hippocampus

(Supplementary Figure 1A, E and I).

Under light microscopy, we were unable to identify any apparent protein aggregation

comparable to that suggested by our and other in-vitro studies of Arx mutant proteins

in Arx(GCG)7

and Arx432-455dup24

embryos (12-14). We analysed over 800 Arx positive

cells, with ~60% from GEs, ~30% from VTh and 10% from dorsal telencephalon,

from 3 independent brain samples per Arx(GCG)7

and Arx432-455dup24

genotype (n=3)

(Figure 2A, A’, C, C’, E and E’). Normal expression pattern was also observed in Arx

positive cells within the cerebral cortex (CCx) and olfactory bulbs (OB) of two

18.5dpc Arx(GCG)7/Y

brains (data not shown). Together these data suggest that the

Arx(GCG)7

and Arx432-455dup24

proteins do not form apparent aggregates in vivo.



7

Reduction of Arx protein abundance in Arx(GCG)7/Y

and Arx 432-455dup24/Y

mutant

brains during neurogenesis.

We noticed a marked reduction of the Arx protein abundance within the developing

telencephalon of the 12.5dpc Arx(GCG)7/Y

and Arx432-455dup24/Y

embryos by

immunofluorescence, including both GEs, VTh (Figure 1 C and E; and

Supplementary Figure 1 C, C’ G and G’) (n=3) and the presumptive cortex (data not

shown). Semi-quantitative Western immunoblot demonstrated a marked reduction of

Arx protein in Arx(GCG)7/Y

(16-17%) (n=3) and Arx432-455dup24/Y

(8-50%) (n=3) at

12.5dpc (Figure 3A and 3B showed 2 out of the 3 embryos), a stage of peak neural

proliferation. This loss of Arx protein was maintained at 14.5dpc, a stage of peak

neurogenesis, 18.5dpc and postnatal day 10 (P10) (Figure 3C and 3D showed 1 out of

2 embryos for each time-point). Thus, both the Arx(GCG)7/Y


mice

modelling the polyalanine expansion mutations lead to similar reduction of Arx

protein in the developing brain.

Interestingly, we noticed a significantly lower body mass in postnatal Arx(GCG)7/Y and

Arx432-455dup24/Y

pups from P5 (25 pups for +/Y; 9 pups for Arx(GCG)7/Y

and 18 pups for

Arx432-455dup24/Y

) (Figure 3E and Supplementary Figure 2A-C). No apparent body size

difference was detected between both mutant and WT embryos at 18.5dpc (15

embryos for +/Y; 5 embryos for Arx(GCG)7/Y

and 12 embryos for Arx432-455dup24/Y

)

(Figure 3E and Supplementary Figure 2A), indicating the weight difference is a

consequence of retarded postnatal growth.



8

Reduced Arx protein abundance is independent of cell loss and as intrinsic to the

expanded polyalanine mutations.

To further investigate the mechanism responsible for the loss of polyalanine expanded

Arx protein and its developmental consequences, we focused on the two regions with

the highest WT Arx protein expression and hence, most striking reduction of Arx

polyalanine expanded mutant protein, the GEs and the VTh. Despite some slight

tissue disorganisation, both Arx(GCG)7/Y


brains showed comparable

mass and cellular density by Haematoxylin and Eosin stains in VTh and GEs where

Arx protein expression is high (n=3) (Supplementary Figure 3A-F and A’-F’). Cell

count analysis also suggested that cell densities were invariable in the respective

regions of LGE and MGE between +/Y (WT) and Arx(GCG)7/Y

(n=3) and Arxdup432-

455dup24/Y embryos (n=3) (MGE and LGE: Figure 3F and G; VTh: data not shown).

These data indicate that the reduction of Arx protein abundance in both Arx(GCG)7/Y

and Arxdup432-455dup24/Y

12.5dpc mouse brains was primarily due to the presence of

polyalanine mutations rather than a secondary consequence of cell or tissue loss.

Loss of Arx protein abundance is not due to the reduction of ArxmRNA

expression.

We found no significant difference in Arx quantity between WT (+/Y) and the two

mutants ( Arx(GCG)7/Y


) in 12.5dpc brains by semi-quantitative RT-

PCR and RT-qPCR (n=4, Figure 4A and B) analysis, indicating no overall loss of Arx

transcript. RNA toxicity from CAG-trinucleotide repeats has been well characterised

in polyglutamine diseases (16). Potential cellular toxicity of polyalanine encoding

GCG-trinucleotide repeats is unknown. In-situ hybridisation showed normal Arx

spatial expression pattern within the 12.5dpc forebrain including the dorsal



9

telencephalon, MGE, LGE and VTh from both Arx(GCG)7/Y


embryos (n=2, Figure 4C-F and J-L). Together, these data suggest the reduction in

Arx protein levels in both mutant models is unlikely to be due to the loss of Arx

mRNA or Arx positive brain domains, but rather reflects a translational and/or post-

translational event.

Differential regulation of Arx transcriptional targets in the Arx(GCG)7/Y

and Arx 432-

455dup24/Y embryonic brains

Arx is well known to be a transcriptional repressor (3, 13, 14, 17, 18). We examined

the impact on transcriptional regulation of characterised ( Lmo1, Shox2, Ebf3, Kdm5c)

and candidate (Gria1, Rab39b and Pax6) Arx target genes by Arx(GCG)7/Y

and Arx432-

455dup24/Y mutations. We undertook RT-qPCR analysis from 12.5dpc whole brains and

telencephalic vesicles (including both the pallium and the subpallium) of the 14.5dpc

and 18.5dpc WT and mutant embryos.

There was a significant increase in Lmo1 expression within Arx(GCG)7/Y

12.5dpc whole

brain (n=4) (Figure 5A) in comparison to that of Arx432-455dup24/Y

or WT. In contrast,

no significant difference in Lmo1 expression was observed between WT and Arx432-

455dup24/Y . At 14.5dpc and 18.5dpc, both mutant mouse models demonstrated a

significantly higher than WT level of Lmo1 expression (Figure 5B and C).

Interestingly, the loss of Lmo1 repression was more severe in Arx(GCG)7/Y

than in

Arx432-455dup24/Y

in 14.5dpc embryos during neural development (Figure 5A and 5B). In

contrast, we found no overall changes in the expression levels of Gria1, Ebf3, Pax6,

Kdm5c and Rab39b between WT and either mutant at any time-point tested (Figure

5A-C). We noted a small but significant reduction in Shox2 expression only when



10

comparing Arx432-455dup24/Y

mice to Arx(GCG)7/Y

mice at 12.5dpc (n=4). However, we

were not able to amplify Shox2 from WT or mutant telencephalic vesicles of 14.5dpc

and 18.5dpc embryos (Figure 5D and data not shown), suggesting the repression on

Shox2 expression by Arx was intact in the telencephalic vesicles of 14.5dpc and

18.5dpc Arx(GCG)7/Y


embryos.

Loss of Lmo1 expression boundaries in both polyalanine mutant mouse models

We performed in-situ hybridisation of Lmo1 in 12.5dpc brain sections from WT,

Arx(GCG)7/Y


embryos to test whether there is an ectopic expression

of Lmo1 in cells that were normally deprived of its expression. Lmo1 expression was

restricted to the VZ of the VTh in WT and both mutants (Figure 4H and I). A diffuse

band of Lmo1 expression was observed in the presumptive cortex of WT 12.5dpc

embryos (Figure 5M and Supplementary Figure 4A-C), with a similar expression

pattern present in both mutant mouse models (Figure 5N-O and Supplementary Figure

4D-E). In contrast, ectopic Lmo1 expression was present within the MZ of MGE, and,

at a much less prominent level, within the MZ of LGE of 12.5dpc Arx(GCG)7/Y

embryos

(n=2, Figure 4N). Despite the lack of significance in RT-qPCR, this expansion of

Lmo1 spatial expression pattern was also observed in the Arx432-455dup24/Y

embryos

(n=2, Figure 4O). Hence, our in-situ study further supported our RT-qPCR data of a

loss of repression of Lmo1 expression in Arx(GCG)7/Y


embryos.

Previous literature (19) and our data indicates that normal Lmo1 expression is

exclusive to the VZ, where neural precursors of MGE and LGE reside. Next, we

asked whether mutant cells with ectopic Lmo1 expression display some progenitor

aspects similar to the VZ precursors (Figure 4P). Nestin is an intermediate filament



11

found mainly in neural progenitors and Phosphohistone-3 is a protein highly

associated with mitotic cycles. No apparent differences in the density, length or

orientation of Nestin filaments were found between either mutant or WT embryos

(data not shown). Similarly, the number of Phosphohistone-3 positive cells within the

MGE were comparable across WT and mutant embryos (data not shown). Together,

these data suggest that cells ectopically expressing Lmo1 were not mitotically active

nor expressing the progenitor filaments common with the VZ precursors.

Discussion

Arx expression can be detected in the developing mouse brain as early as 9.5dpc (20)

suggesting a role in early neural development. Its expression then peaks in the

forebrain, in particular within the subpallium at 12.5 to 14.5 dpc, which are timepoints

critical for neural proliferation and neurogenesis. This Arx expression pattern persists

until the end of embryogenesis and is downregulated during postnatal development

(20, 21). Hence, we focused our analysis during peak neural proliferation and

neurogenesis initiation at 12.5dpc. Our findings of an absence of apparent in-vivo

protein aggregation, but an intrinsic and cell-autonomous loss of polyalanine

expanded Arx protein abundance are in accordance with the previous report (21). We

did not find any difference in the capacity of our Arx antibody to detect WT or

polyalanine expanded Arx proteins when expressed in a range of in-vitro cell lines,

including those with Arx protein aggregations (data not shown). Hence, the difference

in protein abundance detected between the WT and mutant mouse models is unlikely

to be a result of biased antibody affinity towards the WT Arx. Our in-vivo data are in

contrast with previous in vitro analyses (13, 14) showing formation of polyalanine

expanded and length dependant ARX protein aggregates. This discrepancy may have



12

at least two non-mutually exclusive explanations. First, the formation of aggregates

may be dependent on protein abundance such that a physiological level of in-vivo

protein may not be sufficient to induce protein aggregation. Alternatively, it is

plausible that polyalanine expansion mutations result in mis-folded protein and

transient protein aggregates that are efficiently removed by in vivo cellular

machineries, i.e. the proteasome complex. The latter would explain our observation of

reduced Arx protein abundance in both mutant mouse models. In fact, in vivo protein

reduction is common across a number of mice modelling polyalanine expansion

mutations, i.e. Hoxa13 +10Ala , Hoxd13 +7Ala and Sox3 +12Ala (22-24). Together

these data suggest that the loss of protein, but unlikely mRNA, may be a common in

vivo pathogenic hallmark of different by polyalanine expansion mutations in different

genes.

To explore if there is a molecular explanation for the phenotypic variation between

the Arx(GCG)7/Y


mice (6), we speculated that the phenotypic

differences were a result of differential Arx target gene regulation between the two

mutations. To test this we have selected a subset of known and candidate Arx target

genes. Lmo1, Shox2 and Ebf3 are well characterised Arx target genes repressed by Arx

in which regulation on these genes is compromised in the absence of Arx (17, 18).

Recent studies have demonstrated the binding of Arx to the regulatory elements of

many other genes, among these genes previously implicated in epilepsy and

intellectual disability, e.g. KDM5C, GRIA1 and RAB39B (25, 26). In particular,

activation of KDM5C transcription activity by ARX appears to be reduced by the

introduction of polyalanine expansion mutations in-vitro (25). While Arx and Pax4

mutually regulate each other in the specification of glucagon-producing and insulin-



13

producing cells during pancreatic development (27), upregulation of Arx expression

has been observed in the loss of forebrain Pax6 function, a Pax4 brain paralogue, in

both mouse and zebrafish (28, 29). Thus, we speculated that the Pax6 may retain

similar interaction with Arx during brain development. We interrogated the

transcriptional regulation of these selected well known and candidate Arx target genes

(18, 26). Out of the seven genes selected, we detected a more severe loss of Lmo1

repression in Arx(GCG)7/Y

in comparison to Arx432-455dup24/Y

during peak neural

proliferation (12.5dpc) and neurogenesis (14.5dpc), but were equally affected at

18.5dpc when neurogenesis subsides. The temporal and quantitative Lmo1 expression

differences, as seen in these mice models are reminiscent of the differences in clinical

presentations and ARX patients with the respective mutations. We did not identify

any significant changes in the transcriptional regulation of the other six genes in either

mouse model. We reason the lack of in vivo support for these expression differences

might be due to the in-vitro settings of previous studies (25, 30) or due to the temporal

or cellular differences. In fact, a loss of Shox2 and Ebf3, but not Lmo1 repression by

luciferase reporter in in vitro neurons differentiated from transfected embryonic stem

cells with +8Ala in Arx polyalanine tract 1 has been reported by others (30). It is

plausible that this discrepancy is a result of different Arx protein abundance in

transfected in vitro differentiated neurons (30) versus in-vivo neural progenitors in our

study. Otherwise, this difference may be a result of the disparity in the addition of 8

alanine residues in the transfected Arx mutant in-vitro, but only an addition of 7

alanines in the Arx(GCG)7

mouse model.



14

The most plausible explanation for the differential regulation of Lmo1 activity

between the two Arx mutant mouse models, at least in our view, could be the

difference in the amount of the Arx protein loss (16-17% in Arx(GCG)7/Y

versus 8-50%

in Arx432-455dup24/Y

12.5dpc brains), which suggests a different, partial quantitative (and

thus variable) loss of function. Moreover, similar ectopic Lmo1 expression pattern has

been reported in the subpallium of 14.5dpc and 18.5dpc Arx deficient mice, further

supporting this proposition (17, 18). Similarly for Hoxa13 and Sox3, the partial loss of

function secondary to protein reduction is proposed to be responsible for most of the

phenotypic outcomes of the polyalanine expansion mutations due to an overlapping

phenotype spectrum between the null and polyalanine expansion mutant mouse

models (22, 23). Nevertheless, since Lmo1 is directly repressed by Arx (18), the in-

vivo primary impact of polyalanine expansion mutations on transcription regulation

by residual Arx should not be overlooked. In particular, both Arx(GCG)7/Y

and Arx432-

455dup24/Y mice displayed intact Shox2 and Ebf3 repression, indicating normal residual

functions of these mutant proteins in the context of those genes (17, 18).

In vitro studies have readily demonstrated that peptides with >15 alanine residues

have higher propensity to form stable macromolecular β-sheets (31, 32) and alter

molecular folding. The functional disparity between Arx(GCG)7

and Arx432-455dup24

mutation could be a consequence of the positional difference between the two

polyalanine expansion mutations, resulting in distinct conformation changes within

the Arx protein, affecting the folding of different protein domains and ultimately their

function. In fact, functional alteration by polyalanine expansion mutations in

transcription factor Zic2 and Hoxd13 has been proposed as the main contributor to the

pathogenic mechanisms (24, 33). Thus, it is likely that the impact of Arx target gene



15

de-regulation in Arx(GCG)7/Y


mice is a combinatorial outcome of a

partial loss of function, possibly secondary to protein reduction, and an altered

function of the residual mutant Arx protein.

The ectopic Lmo1 expression strongly suggests that those cells within the MZ of the

MGE and LGE (mostly maturing interneurons) already have a change of “identity” by

12.5dpc in both mutant mouse models. However, our evidence of Nestin and

Phosphohistone-3 expression suggested they do not share these two progenitor

properties with the VZ precursor cells. Further studies will need to establish their

lineages and the identity of these “supposing” maturing interneurons and the impact

of ectopic Lmo1 on their cellular fates.

Despite LMO1 being a well-studied oncogene that promotes proliferation in

neuroblastoma cells and maintenance of self-renewal in T-cell lymphoblastic

leukaemia (34, 35), very little is known regarding a role in neural development. Given

the function of LMO1 in cancer cells, and the restricted forebrain expression within

the VZ, where proliferative neural progenitors reside, it would be reasonable to

speculate that Lmo1 may have a pro-progenitor property in neural development. Lmo3

is a highly conserved and functionally redundant paralogue of Lmo1 and is directly

bound by Arx within its regulatory region in 15.5dpc mouse brain and neuroblastoma

cells (26, 36). Moreover, Lmo1, Lmo3 and Lmo4, a highly conserved Lmo family

member important for neurulation (37), were all ectopically expressed within the

subpallium of 14.5 Arx deficient mice (17). It will be of interest to investigate if Lmo3

and Lmo4 are also ectopically expressed within the MGE and LGE of the Arx(GCG)7

and Arx432-455dup24

mouse models.



16

Arx(GCG)7

mutant mice displayed morphologically normal CCx but mild interneuron

deficiency from the subpallium (6). Unlike the MGE and LGE, we found Lmo1

expression pattern was intact within the dorsal telencephalon of both mutant models.

This observation is in agreement with a study demonstrating the residual function of

Arx(GCG)7

mutant protein was sufficient for a near complete rescue of cortical

proliferation and migration in an Arx null mouse model (30). Similarly, there was no

ectopic Lmo1 expression in the MZ of the VTh. Together, our results support the

observation of a dynamic role for Arx in regulating target genes during dorsal

(developing cortex) and ventral (GEs and VTh) forebrain development (30, 38).

Interestingly, a recent study has shown that Arx is required for the patterning and

development of dopaminergic neurons in zebrafish VTh (39). Future work will need

to address whether this role of Arx in the VTh is conserved in mammals and how

polyalanine expansion mutations affect the development of this domain.

Polyalanine expansion mutations account for over half of mutations found in patients

with ARX mutations. Unlike mutations that cause brain malformations, patients with

polyalanine expansion mutations showed no gross morphological defects and suffer

from varying degrees of ID with and without epilepsy (3). However, the molecular

mechanism that underlies the pathogenesis of these polyalanine expansion mutations

is not well understood. Here, we undertook comparative expression analysis of the

mouse lines modelling the two most common ARX polyalanine expansion mutations.

Firstly, we did not find any support for in vivo Arx mutant protein aggregation in two

mouse models studied. Secondly, we demonstrated a reduction of Arx protein

abundance without loss of cells in both mutant mouse models, including postnatal



17

growth retardation. We have identified a specific impact on Lmo1 gene expression in

mutant mouse models. Overall our data leads us to propose a region- and mutation-

specific loss of Lmo1 transcription regulation as one of the contributors to the more

severe phenotypic symptoms observed in Arx(GCG)7/Y

mice compared to Arx432-455dup24

mice, and possibly human patients with the equivalent mutations.

Material and methods

Animals and tissue collection

All animal procedures were approved by the Animal Ethics committee of the

University of Adelaide, the SA Pathology Animal Ethics committee and the Animal

Ethics committee of the Women’s and Children’s Hospital, Adelaide. Five ArxGCG7/+

(BRC number: 03654) and four Arx432-455dup/+

(BRC number: 03653) heterozygote

females were imported from RIKEN Bioresource Centre, Japan (6). Both mouse

strains were maintained in C57BL/6 background. Pregnant dams were euthanized by

cervical dislocation followed by decapitation of pups or embryos. For RNA and

protein extraction, tissue were snap frozen at -80°C. For frozen tissue sections, whole

mount tissue was prepared as described previously (40) and sectioned at 10μM thick

using Microm HM505E.

Genotyping

Genotyping gDNA was extracted as per Maxwell® 16 Tissue DNA purification Kit

manual (Promega). Genotyping PCR was performed using FailSafe™ PCR 2X

PreMix J (Epicentre) as follows: 35 cycles of 30sec of 94°C for denaturation, 30sec of



18

60°C for annealing and 40sec of 72°C for elongation. Primers to amplify the Arx

knock-in region were described in (6). We also included Sry sexing PCR as part of

our genotyping pipeline (Sequences in Supplementary Table 1).

Immunofluorescence

Prior to Arx immunofluorescence, frozen tissue sections were air-dried for 1 hour at

room temperature. All procedures were performed in a humidified chamber to prevent

drying of tissue sections. Tissue sections were permeabilised in 1xPBS + 0.5% Triton

for 5 minutes, blocked with blocking solution (10% horse serum and 10% BSA in

1xPBS + 0.1% Triton) for one hour at room temperature. Sections were then

incubated in primary Arx antibody (rabbit anti-ARX at 1/500, (20, 21)) in blocking

solution at 4°C overnight. Sections were washed in 1xPBS + 0.01%Tween 20 for 10

minutes 3 times. They were then incubated at in 1/400 donkey anti-rabbit IgG

Alexa488 secondary antibody (Life Technologies) at room temperature for 4 hours.

Sections were washed again in 1xPBS + 0.01%Tween 20 for 10 minutes 3 times.

prior to be immediately mounted with ProLong Gold Antifade Reagent with DAPI

(Life Technologies). Mounted sections were allow to cure in the dark at room

temperature overnight prior to analysis and further storage at 4°C in the dark.

Image analysis

All images were analyzed using Olympus IX81 inverted microscope equipped with

CellSens 1.3 Software. Immunofluorescence images were acquired by Olympus

XM10 black and white camera, while bright field images were captured using

Olympus DP70 digital colour camera. All captured images were processed by Adobe

Photoshop CS5.



19

Semi-quantitative RT-PCR and RT-qPCR

Collected tissue were homogenised with 21G needles and total RNA was extracted

using Trizol (Invitrogen) and RNeasy Mini Kit (Qiagen) and treated with DNase I

(Qiagen) according to the manufacturer’s instruction. cDNA was prepared as

described in SuperScript III reverse transcriptase (Invitrogen) manual with 1 g of

RNA primed by random hexanucleotides. Cycling conditions for RT-PCR are similar

to the above genotyping protocols. RT-qPCR was performed as described previously

(40). No template and no reverse transcriptase controls were included for product

specificity. For Lmo1, Shox2 and Ebf3, reactions were prepared as described in

Taqman® PreAmp Master Mix Kit user guide (Applied Biosystem). Expression

values were normalised to reference gene Gapdh. Taqman® assays ID are:

Mm00475438_m1 FAM ( Lmo1), Mm00443183_m1 FAM (Shox2), Mm00438637

FAM ( Ebf3) and Mm99999915_g1 VIC (Gapdh). For Arx, Gria1, Kdm5c, Pax6 as

well as Lmo1, reactions were setup as described in SYBR® Green PCR Master Mix

and RT-PCR Reagents Kit user guide (Applied Biosystem). Melt curve analysis was

performed to ensure amplification efficiency. Expression values were normalised to

reference gene Sdha. Analyses of Lmo1 expression at 12.5dpc and 14.5dpc time-point

were performed using both SYBR® and Taqman® assay. Comparable results were

obtained between the two assays while only values from SYBR® assay was presented

in the result section. Primers listed in Supplementary Table 1.



20

Protein extraction, SDS-PAGE and Western Blot

Dissected tissue was homogenised by 21G needles prior cell lysates extraction for

Western immunoblot as described previously (14, 41). Primary antibodies used were:

rabbit anti-Arx (1/700, (21)); and mouse anti-Actb (1/2000, Sigma Aldrich).

Secondary antibodies used are: goat anti-rabbit IgG HRP (Dako); and goat anti-mouse

IgG HRP (Dako). To quantify Arx protein abundance, the intensity histogram for

each band/sample was obtained by ImageJ. Signal intensity was calculated by the area

under the signal peak for each histogram. All values were normalised to WT samples

within each blot. Percentages of Arx protein abundance were derived from normalised

values.

Haematoxylin and Eosin stains

Haematoxylin and Eosin stains were performed as previously (40).

In- situ hybridisation

Protocols are as performed previously (40). Arx and Lmo1 sequences were amplified

from cDNA cloned into pGEM®T Vector (Promega) prior to riboprobe synthesis.

(See Supplementary Table 1 for primers). Riboprobes were synthesized by T7 after

linearization with SpeI.

Cell counts

Cell counts were performed on DAPI stained nuclei. Cell density analysis was only

performed on sections of anatomically comparable planes. For each anatomical

domain, there is a high-density zone (made up of regions mostly with proliferating



21

cells) and a low-density zone (made up with regions with cells that are low or have no

proliferation). Each zone was arbitrarily divided into four bins of equal area. Cell

count was performed twice in each bin as number of cells/mm2 and an average was

taken. Then, the cell count of a zone was calculated by averaging the cell counts of

the four bins. A schematic diagram of with zoning and binning strategy was presented

as Figure 3F-G in result section.

Statistical analyses

To perform statistical analyses for postnatal weight, cell counts and gene expression,

the median and number of observations were given to the three groups, i.e. +/ Y,

Arx(GCG)7/Y


. A Kruskal-Wallis test was performed to establish

whether there were any significant differences between +/ Y, Arx(GCG)7/Y

and Arx432-

455dup24/ Y. If a significant difference were found amongst all groups, pair-wise

comparisons by Kruskal-Wallis tests were then carried out to determine the

significance between each paired group.



22

Acknowledgements

This work was supported by grants from the National Health and Medical Research

Council of Australia (Project Grant 1002732 to C.S.; Principle Research Fellowship

508043 to J.G.) and Australian Research Council (Future Fellowship FT 120100086

to CS). We would like to thank Laboratory Animal Services Facility at the Women’s

and Children’s Hospital (Adelaide) for their kind assistance. We appreciate the

statistical help from Mr Thomas Sullivan, Ms Nancy Briggs and Ms Michelle Lorimer

from Data Management and Analysis Centre at the University of Adelaide. We would

like to thank Dr K. Kitamura, Ms M. Yanazawa and Riken BRC and Mitsubishi

Chemical Corporation for kindly providing Arx(GCG)7/Y

and Arx432-455dup24/ Y KI

mice.

Conflict of Interest Statement

The Authors declare that there is no conflict of interest.



23

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27

Legends to Figures

Figure 1: Arx protein localisation was restricted to the developing forebrain at

12.5dpc. A and E: WT Arx was localised at the SVZ and the MZ of MGE and LGE.

Note that Arx protein levels were higher at the MZ of both regions and was the

highest in migrating neurons at the very dorsolateral edge of MGE and LGE (#). C

and G: A markedly reduced level of Arx protein was observed in both Arx(GCG)7/Y

and

Arx432-455dup24/Y

embryonic brains, noticeably at the dorsolateral edge of MGE and

LGE where WT protein levels were higher. Albeit with lower intensity, Arx was still

detected in cells within other regions of the MGE and LGE (C and G). The broken

lines divide the VZ from the SVZ and the MZ. (B), (D), (F) and (H) are DAPI stains

of (A), (C), (E) and (G) respectively. Coronal sections shown with top to bottom as

dorsal to ventral. Scale bar: 200µM (A-H).

Figure 2: An absence of in-vivo protein aggregation was observed in Arx+ cells

within the 12.5dpc developing telencephalon in both Arx(GCG)7/Y and Arx 432-

455dup24/Y mouse models. A, C and E: An example of Arx in migrating cells at the

dorsolateral GEs, where WT Arx protein signal is strong. A’, C’ and E’: A punctate

localisation pattern was observed in all samples studied. (A’), (C’) and (E’) are

magnified boxed regions of (A), (C) and (E) respectively. (B), (D) and (F) are DAPI

stains of the same respective section in (A), (C) and (E). Coronal sections shown with

top to bottom as dorsal to ventral. Scale bar: 12.5 µM (A-F); and 6.25µM (A’, C’ and

F’).



28

Figure 3: Arx(GCG)7/Y

and Arx 432-455dup24/Y

mouse models displayed a marked

reduction in protein abundance in 12.5dpc embryonic brains and significantly

lower postnatal body mass. A and B: Western immunoblots showing consistent Arx

protein abundance across WT 12.5dpc brains (A). Lower protein abundance was

observed in both Arx(GCG)7/Y


(B). C and D: Western immunoblots

showing Arx protein levels decreasing from peak at embryonic 12.5, 14.5 and

18.5dpc brain to a lower level at P10 (C). In Arx(GCG)7/Y


mutants,

Arx protein reduction persisted from early neurogenesis at 12.5dpc into postnatal

development at P10, which was almost undetectable (D). E: Although Arx(GCG)7/Y

,

Arx432-455dup24/Y and WT (+/Y) embryos have similar body weight at 18.5dpc, a

significantly lower body mass in both mutant pups was evident 5 days later (P5). F

and G: Comparable cellular density was observed between the respective high-

density (High) and low-density zone (Low) of the LGE and MGE in 12.5dpc WT and

mutant embryos (G) with a schematic diagram showing the strategy for cellular

quantification (F). Each brain domain was divided into two zones: high cellular

density (above broken lines, mostly made up of VZ and SVZ and some dorsal MZ)

and low cellular density (below broken lines, mostly made up of ventral MZ). Each

zone was further divided into four bins (yellow boxes) for quantification. Coronal

section shown with top to bottom as dorsal to ventral. CB: Postnatal day 9 cerebellum

was used as a control deprived of Arx protein expression. * p<0.005; **p<0.0001.

Scale bar: 400µM.

Figure 4: The expression pattern of Arx, but not, Lmo1, was intact in both

Arx(GCG)7/Y


12.5dpc embryos . A and B: Arx transcript level was

comparable across WT and both mutants by semi-quantitative RT-PCR (A) or RT-



29

qPCR (B). C-F: Arx mRNA expression was maintained within the VTh (in black

broken lines) of both mutants, with cells immediately neighbouring the VZ (asterisk,

immediately right of the white broken line) showing more intense expression. G-I: In

contrast, Lmo1 was expressed within the proliferating cells at the VZ (left to the white

broken line) of the VTh (enclosed in black broken lines) in WT and both mutants. J-

L: Arx expression domains were maintained in both mutants, with its expression

mainly in the MZ (below the white broken line) and migrating cells (“plus” signs) of

the MGE and LGE. A lower but detectable level of Arx was present at the dorsal

telencephalon (presumptive cortex, white arrowheads). M-O: Lmo1 expression was

mutually exclusive to that of ARX and restricted to the VZ (above the white broken

line) in both WT MGE and LGE (M). Expansion of Lmo1 expression (black

arrowheads) into the MZ (below the white broken line) was apparent, particularly in

both mutant MGEs. In contrast, the diffuse and weak Lmo1 expression pattern in the

dorsal telencephalon appeared to be intact in both mutant mouse models (black

arrows, see Supplementary Figure 4 for magnified region of the dorsal telencephalon).

P: Schematic diagram showing the migrating routes of neural progenitors and

interneurons during development. Note that Lmo1 labels progenitor cells while Arx

labels mostly postmitotic interneurons. Coronal sections shown with top to bottom as

dorsal to ventral. CB: Postnatal day 9 cerebellum was used as a control deprived of

Arx expression. Scale bar: 250µM (C-I); 500µM (J-O).

Figure 5: Loss of Lmo1 repression by Arx in mutant Arx(GCG)7/Y

and Arx 432-

455dup24/Y embryos at various time-points. A: There was a significant increase in

Lmo1 mRNA expression within the brain of 12.5dpc Arx(GCG)7/Y

(~1.5-fold) but not

Arx432-455dup24/Y

. The expression level of Rab39b, Pax6 , Gria1, Ebf3, Shox2 and



30

Kdm5c were relatively similar between WT and either of the mutants. B and C: The

expression of Lmo1 was elevated within the 14.5dpc and 18.5dpc telencephalic

vesicles of both Arx(GCG)7/Y


, while Rab39b, Pax6 , Gria1, Ebf3 and

Kdm5c showed similar expression level within the telencephalic vesicles of WT and

both mutant embryos. Note a significantly higher Lmo1 expression level was detected

in Arx(GCG)7/Y

when compared to Arx432-455dup24/Y

in 14.5dpc embryos. D: Shox2

expression was not detected within the 14.5dpc telencephalic vesicles, but was present

within brain regions caudal to the telencephalon, possibly the VTh. Relative

expression of genes was normalised to the expression of the reference genes, Sdha or

Gapdh. Relative gene expression for Arx(GCG)7/Y and Arx432-455dup24/Y mutant embryos is

presented in comparison to WT, which is defined as 1. *p<0.05; **p<0.005.



31

Abbreviations

CCx cerebral cortex

dpc days post-coitum

ID intellectual disability

GE ganglionic eminence(s)

HTh hypothalamus

LGE lateral ganglionic eminence

LV lateral ventricles

MGE medial ganglionic eminence

MZ mantle zone

OB olfactory bulb(s)

P postnatal day

VTh ventral thalamus

VZ ventricular zone

WT wildtype

XLID X-linked intellectual disability



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33



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35



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hum. mol. genet. 2013 lee hmg ddt503

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