Aristaless-related Homeobox Research Articles

Estradiol does not affect spasms in the betamethasone-NMDA rat model of infantile spasms.

This study attempted to validate the effects of neonatal estradiol in ameliorating the spasms in the prenatally betamethasone-primed N-methyl-d-aspartate (NMDA) model of infantile spasms in rats as shown previously in a mouse Arx gene knock-in expansion model of infantile spasms. Neonatal rats prenatally exposed to betamethasone (on day 15 of pregnancy) were treated with subcutaneous 40 ng/g estradiol benzoate (EB) between postnatal days (P)3-P10 or P0-P5. A synthetic estrogen analogue, diethylstilbestrol, was used between P0 and P5 (2 μg per rat, s.c.). On P12, P13, and P15, the rats were subjected to NMDA-triggered spasms, and latency to onset and number of spasms were evaluated. Rats with EB on P3-P10 were tested after spasms in the open field, novel object recognition, and elevated plus maze to determine effects of treatment on behavior. Additional rats with P3-P10 or P0-P5 EB were investigated for γ-aminobutyric acid (GABA)ergic neurons (glutamate decarboxylase [GAD]67 expression) in the neocortex. As a positive control, a group of rats received either subcutaneous adrenocorticotropic hormone (ACTH) (2 × 0.3 mg/kg on P12 and 3 × 0.3 mg/kg on P13 and P14) or vehicle after the first episode of spasms on P12. Neither EB treatment nor diethylstilbestrol consistently affected expression of spasms in this model, although we found a significant increase in GAD67-immunopositive cells in the neocortex after P3-P10 and P0-P5 EB treatment, consistent with a study in mice. Behavioral tests showed increase in lateralization in male rats treated with P3-P10 EB, a behavioral trait usually associated with female sex. Diethylstilbestrol treatment in male rats resulted in arrested pubertal descent of testes. ACTH had robust effects in suppressing spasms. Treatment of infantile spasms (IS) using neonatal EB may be justified in those cases of IS that present with detectable deficits in GABAergic neurons. In other types of IS, the efficacy of neonatal EB and its analogues is not supported.

Developmental interneuron subtype deficits after targeted loss of Arx

BackgroundAristaless-related homeobox (ARX) is a paired-like homeodomain transcription factor that functions primarily as a transcriptional repressor and has been implicated in neocortical interneuron specification and migration. Given the role interneurons appear to play in numerous human conditions including those associated with ARX mutations, it is essential to understand the consequences of mutations in this gene on neocortical interneurons. Previous studies have examined the effect of germline loss of Arx, or targeted mutations in Arx, on interneuron development. We now present the effect of conditional loss of Arx on interneuron development.ResultsTo further elucidate the role of Arx in forebrain development we performed a series of anatomical and developmental studies to determine the effect of conditional loss of Arx specifically from developing interneurons in the neocortex and hippocampus. Analysis and cell counts were performed from mouse brains using immunohistochemical and in situ hybridization assays at 4 times points across development. Our data indicate that early in development, instead of a loss of ventral precursors, there is a shift of these precursors to more ventral locations, a deficit that persists in the adult nervous system. The result of this developmental shift is a reduced number of interneurons (all subtypes) at early postnatal and later time periods. In addition, we find that X inactivation is stochastic, and occurs at the level of the neural progenitors.ConclusionThese data provide further support that the role of Arx in interneuron development is to direct appropriate migration of ventral neuronal precursors into the dorsal cortex and that the loss of Arx results in a failure of interneurons to reach the cortex and thus a deficiency in interneurons.

Research Resource: Genetic Labeling of Human Islet Alpha Cells.

The 2 most abundant human pancreatic islet cell types are insulin-producing β-cells and glucagon-producing α-cells. Defined cis-regulatory elements from rodent Insulin genes have permitted genetic labeling of human islet β-cells, enabling lineage tracing and generation of human β-cell lines, but analogous elements for genetically labeling human α-cells with high specificity do not yet exist. To identify genetic elements that specifically direct reporter expression to human α-cells, we investigated noncoding sequences adjacent to the human GLUCAGON and ARX genes, which are expressed in islet α-cells. Elements with high evolutionary conservation were cloned into lentiviral vectors to direct fluorescent reporter expression in primary human islets. Based on the specificity of reporter expression for α- and β-cells, we found that rat glucagon promoter was not specific for human α-cells but that addition of human GLUCAGON untranslated region sequences substantially enhanced specificity of labeling in both cultured and transplanted islets to a degree not previously reported, to our knowledge. Specific transgene expression from these cis-regulatory sequences in human α-cells should enable targeted genetic modification and lineage tracing.

The mammal-specific Pdx1 Area II enhancer has multiple essential functions in early endocrine cell specification and postnatal β-cell maturation.

The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5' conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (-2139 to -1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo Pdx1ΔAREAII/- mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions.

Can the p.Thr1174Ser Mutation in SCN1A Gene Shape Genetic Background in Epileptic Encephalopathies?

Dravet Syndrome (DS) and Genetic Epilepsy with Febrile Seizures (GEFS+) are very often caused by mutations in the SCNA1A gene. These mutations also have been identified in families with migraine phenotypes, supporting the link between migraine and epilepsy. The SCN1A substitution p.Trp1174Ser has been reported as a cause of familial migraine and familial mixed phenotypes with seizures / hemiplegic migraine. We present this mutation as a causative factor of familial GEFS+ syndrome, but also as a factor potentially changing the phenotypes of the epileptic encephalopathies caused by mutations in the SCN1A, ARX or PCDH19 genes. Substitution p.Trp1174Ser was identified in five probands clinically diagnosed as spectrum of GEFS+ or DS. As it has not been regarded as significant for the epileptic encephalopathy, they underwent additional testing according to the revised phenotypes. Probands were finally diagnosed with GEFS+ (p.Trp1174Ser SCN1A mutation only) and epileptic encephalopathies: DS (p.Arg712* and p.Arg1245* in SCN1A), Epilepsy and Mental Retardation Limited to Females (p.Asp155Tyr in PCDH19) and atypical West Syndrome (del79nt IVS4/Ex5 in ARX). This study indicates a complex involvement of some SCN1A mutations in epilepsies / epileptic encephalopathies also as a modifying factor with the SCN1A, PCDH19, ARX and possibly mutations in other genes. In cases with atypical or plus course or more severe course the possible involvement of other genetic factors should always be considered. Additional modifiers identification may influence on clinical prognosis, patient management and genetic counselling.

The Role of ARX in Human Pancreatic Endocrine Specification.

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

ARX and SHH gene mutation analyses revealed no mutation in patients with agenesis/dysgenesis of the corpus callosum

Mutations in sonic hedgehog (SHH; OMIM *600725) and the aristaless-related homeobox gene (ARX; OMIM *300382) can both result in severe brain malformation. Haploinsufficiency of SHH has been shown to cause not only midline structure defects such as holoprosencephaly, corpus callosum agenesis but also developmental delay and/or craniofacial dysmorphic features including microcephaly, hypotelorism, or a single central incisor. Mutations in the ARX gene may result in different phenotypes as well, such as X-linked mental retardation (XLMR; OMIM #300419), X-linked lissencephaly with ambiguous genitalia (XLAG; OMIM #300215), Partington syndrome (OMIM #309510), X-linked myoclonic epilepsy (OMIM #300432), X-linked West syndrome (OMIM #308350), and agenesis of the corpus callosum with abnormal genitalia (OMIM #300004). In a total number of 27 patients with complete or partial absence of the corpus callosum, SHH and ARX genes were investigated in order to evaluate whether mutations in SHH and ARX result in partial (dysgenesis) or complete (agenesis) absence of the corpus callosum. No causative mutations could be detected suggesting that these genes may not play a major role in corpus callosum formation.

ARX and other single genes in X-linked mental retardation: revisiting a population-based study

It has been calculated that monogenic genes for Xlinked mental retardation (XLMR) account for <10% of male MR [1]. XLMR can be classified as syndromic, referring to conditions with distinct neurological, physical or biochemical abnormalities, or nonsyndromic (unspecified or nonspecific). More than 20 genes are known to be associated with syndromic XLMR [2] and 13 genes have so far been identified with nonsyndromic XLMR [3]. The most common XLMR condition, fragile X syndrome (FRAXA) due to mutation in the FMR1 gene, occurs in approximately 1 in 6000 males in various populations, or in 2–2.5% in cohorts of MR males [4]. Although the search for genes has been very active in recent years, mutations in a “major” causative gene underlying a larger proportion of unexplained XLMR remain to be identified. Recently, the identification of mutations in the Aristaless related homeobox gene (ARX) in syndromic as well as non syndromic conditions [5,6] created optimism that ARX could be a major gene in XLMR, particularly since the phenotypic range

Unraveling the pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approach.

The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members.

O-010 Comparative Neuropathology Of Lissencephaly With Arx Mutation: Consideration Of Neocortical Interneuron Distribution

Background X-linked lissencephaly with abnormal genitalia (XLAG) is established as one disease entity. XLAG, showing severe neonatal seizure and developmental delay, is a rare disorder caused by mutations in the aristaless-related homeobox ( ARX ) gene, located in Xp22.13. Arx -null mice for human XLAG model showed loss of tangential migration of GABAergic interneurons. Objectives We investigated subpopulation of GABAergic interneurons in the brain of an infant with XLAG, who had a nonsense mutation of the ARX gene, compared with those of age-matched normal control, Miller-Dieker syndrome (MDS) as a type I lissencephaly, and polymicrogyria of Fukuyama type congenital muscular dystrophy (FCMD) as a type II lissencephaly. Methods We used paraffin-embeded brain tissues of two XLAG, three MDS and four FCMD, with an informed consent of their parents. We performed immunocytochemistry for interneuron and migration markers. Results Glutamic acid decarboxylase (GAD) and calretinin (CR) containing (+) cells were significantly very few in the neocortex and located in the white matter and neocortical subventricular zone. In the neocortical subventricular region, the GAD+ and CR+ cells had Mash -1 protein, like a radial migration marker, and nestin protein. On the contrary, MDS showed relative low concentration of GAD+ cells. FCMD revealed random distribution of these marked cells. Conclusions ARX controls not only tangential migration of GABAergic interneurons from the ganglionic eminence, but also may serve to induce radial migration from the neocortical subventricular zone. MDS and FCMD also demonstrated abnormal distribution of neocortical interneurons, but those severities are different in each type of lissencephaly.

Renzo Guerrini: seeing things other people don't see

Renzo Guerrini: seeing things other people don't see

Conditional Loss of Arx From the Developing Dorsal Telencephalon Results in Behavioral Phenotypes Resembling Mild HumanARXMutations

Mutations in the Aristaless-Related Homeobox (ARX) gene cause structural anomalies of the brain, epilepsy, and neurocognitive deficits in children. During forebrain development, Arx is expressed in both pallial and subpallial progenitor cells. We previously demonstrated that elimination of Arx from subpallial-derived cortical interneurons generates an epilepsy phenotype with features overlapping those seen in patients with ARX mutations. In this report, we have selectively removed Arx from pallial progenitor cells that give rise to the cerebral cortical projection neurons. While no discernable seizure activity was recorded, these mice exhibited a peculiar constellation of behaviors. They are less anxious, less social, and more active when compared with their wild-type littermates. The overall cortical thickness was reduced, and the corpus callosum and anterior commissure were hypoplastic, consistent with a perturbation in cortical connectivity. Taken together, these data suggest that some of the structural and behavioral anomalies, common in patients with ARX mutations, are specifically due to alterations in pallial progenitor function. Furthermore, our data demonstrate that some of the neurobehavioral features found in patients with ARX mutations may not be due to on-going seizures, as is often postulated, given that epilepsy was eliminated as a confounding variable in these behavior analyses.

ArxIs Required for Specification of the Zona Incerta and Reticular Nucleus of the Thalamus

Mutations in the aristaless-related homeobox (ARX) gene result in a spectrum of structural and functional nervous system disorders including lissencephaly, movement disorders, intellectual disabilities, and epilepsy. Some patients also have symptoms indicating hypothalamic dysfunction, but little is known about the role of ARX in diencephalic development. To begin evaluating diencephalic defects, we examined the expression of a panel of known genes and gene products that label specific diencephalic nuclei in 2 different Arx mutant mouse lines at E18.5. Male mice engineered to have a polyalanine expansion mutation (Arx) revealed no expression differences in any diencephalic nucleus when compared with wild-type littermates. In contrast, mice null for Arx (Arx) lost expression of specific markers of the thalamic reticular nucleus and zona incerta (ZI) while retaining expression in other thalamic nuclei and in the hypothalamus. Tyrosine hydroxylase, a marker of the dopaminergic A13 subnucleus of ZI, was among those lost, suggesting a requirement for Arx in normal thalamic reticular nucleus and ZI development and, specifically, for A13 dopaminergic fate. Because the ZI and A13 regions make connections to several hypothalamic nuclei, such misspecification may contribute to the "hypothalamic dysfunction" observed in some patients.

DTI tractography of lissencephaly caused by TUBA1A mutation

Several genetic causes have been identified in lissencephaly, including mutations in LIS1, DCX, ARX, RELN, VLDLR, WDR62, and TUBA1A genes. One may sometimes suggest the most likely gene mutation upon MRI findings when some specific features are seen, although image findings overlap among the causative gene mutations. A 1-year-old boy presented with intractable epilepsy, developmental delay and microcephalus. Brain MRI revealed lissencephaly with severe dysplasia of the cerebellum and brainstem. Diffusion tensor imaging (DTI) visualized radially aligned structures within the cortex, which is not observed in normal brain after birth (Fig. 1). Combination of severe posterior fossa abnormalities with lissencephaly suggested TUBA1A as the most likely responsible gene mutation. Mutation analysis of TUBA1A gene revealed a missense mutation of c.1205G>A, which was negative in both parents indicating a de novo mutation. Fig. 1 T1-weighted axial image (a) and T2-weighted axial images (b) show diffuse thickening of the cortex with lack of sulcation, accompanied by severe dysplasia of the brainstem and cerebellum. Obscuration of the contour of the basal ganglia and internal capsule ... Mutation of TUBA1A gene, coding for alpha I tubulin that constitutes subunit of microtubule, is a relatively rare cause of lissencephaly. The most consistent feature of lissencephaly due to TUBA1A mutation is severe cerebellar and brainstem abnormalities. Other characteristics include dysmorphic basal ganglia with hypoplastic internal capsule, callosal hypoplasia, and microcephaly, as seen in this case [1, 2]. Recently, mutations in the neuronally expressed tubulin genes have been reported to cause a spectrum of overlapping cortical malformations ranging from lissencephaly to polymicrogyria, and categorized as “tubulinopathy” [3]. Although cerebellar abnormalities may be present in LIS1 and DCX mutation, they are much less severe than in TUBA1A mutation. The radially aligned structure within the cortex and increase of cortical fractional anisotropy (FA) are transiently observed in the normal developing brain around 15–28 gestational weeks [4]. Normally, the cortical FA declines with development and approaches zero by 36 weeks [5]. This transient radially aligned anisotropic structure has been presumed to represent the presence of radial glia [4, 6]. The radial glias retract their ventricular and pial attachments and differentiate into astrocytes as the brain matures normally, with corresponding decrease in the cortical FA value. The radially oriented structure within the cortex demonstrated in this case may represent remains of radial glia or persistent radial orientation of neurons arrested in the migration process [7].

Molecular studies on ARX gene in syndromic and non-syndromic mental retardation

Background Mental retardation (MR) is frequently the result of genetic mutations. Syndromic mental retardation is intellectual deficits associated with other medical and behavioral signs and symptoms. Non-syndromic mental retardation refers to intellectual deficits that appear without other abnormalities. The newly identified ARX (Aristaless related homeobox) gene consists of five exons and encodes 562 amino acid proteins and is thought to regulate brain development. Mutations in the ARX gene are associated with a diverse spectrum of phenotypes ranging from severe developmental abnormalities of the brain to syndromic and nonsyndromic forms of X-linked mental retardation (XLMR) syndromes that can be associated with normal or abnormal brain morphology. Mutations in the human ARX gene are the major cause of developmental and neurological disorders.

The c.429_452 duplication of the ARX gene: a unique developmental-model of limb kinetic apraxia

BackgroundThe c.429_452dup24 of the ARX gene is a rare genetic anomaly, leading to X-Linked Intellectual Disability without brain malformation. While in certain cases c.429_452dup24 has been associated with specific clinical patterns such as Partington syndrome, the consequence of this mutation has been also often classified as “non-specific Intellectual Disability”. The present work aims at a more precise description of the clinical features linked to the c.429_452dup24 mutation.MethodsWe clinically reviewed all affected patients identified in France over a five-year period, i.e. 27 patients from 12 different families. Detailed cognitive, behavioural, and motor evaluation, as well as standardized videotaped assessments of oro-lingual and gestural praxis, were performed. In a sub-group of 13 ARX patients, kinematic and MRI studies were further accomplished to better characterize the motor impairment prevalent in the ARX patients group. To ensure that data were specific to the ARX gene mutation and did not result from low-cognitive functioning per se, a group of 27 age- and IQ-matched Down syndrome patients served as control.ResultsNeuropsychological and motor assessment indicated that the c.429_452dup24 mutation constitutes a recognizable clinical syndrome: ARX patients exhibiting Intellectual Disability, without primary motor impairment, but with a very specific upper limb distal motor apraxia associated with a pathognomonic hand-grip. Patients affected with the so-called Partington syndrome, which involves major hand dystonia and orolingual apraxia, exhibit the most severe symptoms of the disorder. The particular “reach and grip” impairment which was observed in all ARX patients, but not in Down syndrome patients, was further characterized by the kinematic data: (i) loss of preference for the index finger when gripping an object, (ii) major impairment of fourth finger deftness, and (iii) a lack of pronation movements. This lack of distal movement coordination exhibited by ARX patients is associated with the loss of independent digital dexterity and is similar to the distortion of individual finger movements and posture observed in Limb Kinetic Apraxia.ConclusionThese findings suggest that the ARX c.429_452dup24 mutation may be a developmental model for Limb Kinetic Apraxia.

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon+ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.

Reduced polyalanine-expanded Arx mutant protein in developing mouse subpallium alters Lmo1 transcriptional regulation

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 Arx(432-455dup24)). 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 with Arx(432-455dup24/Y) mice at 12.5 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 Arx(432-455dup24) mouse models. It is yet to be determined what effect these mutations have on ARX protein in affected males in the human setting.

ARX Regulates Cortical Intermediate Progenitor Cell Expansion and Upper Layer Neuron Formation Through Repression of Cdkn1c

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.