Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.
X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic epilepsy of early childhood with intractable seizures, intellectual disability, and poor prognosis. A spectrum of mutations in the Aristaless-Related Homeobox gene (ARX) has been linked to ISSX, and downstream targets of this interneuron-expressed transcription factor are being defined. Recent advances combining in vitro and in vivo methods have unveiled complex interactions between Arx and its binding partners and their effects on cell migration and maturation that can help explain the diversity of ARX phenotypes. New mutant mouse models of Arx-induced pathology, including a recent human triplet-repeat expansion mutation with a phenotype of infantile spasms and electrographic seizures, provide valuable tools for exploring the pathophysiology of Arx and substrates for testing novel therapies.
Aristaless related homeobox (ARX) is a paired-type homeobox gene. ARX function is frequently affected by naturally occurring mutations. Nonsense mutations, polyalanine tract expansions and missense mutations in ARX cause a range of intellectual disability and epilepsy phenotypes with or without additional features including hand dystonia, lissencephaly, autism or dysarthria. Severe malformation phenotypes, such as X-linked lissencephaly with ambiguous genitalia (XLAG), are frequently observed in individuals with protein truncating or missense mutations clustered in the highly conserved paired-type homeodomain.
We have identified two novel point mutations in the R379 residue of the ARX homeodomain; c.1135C>A, p.R379S in a patient with infantile spasms and intellectual disability and c.1136G>T, p.R379L in a patient with XLAG. We investigated these and other missense mutations (R332P, R332H, R332C, T333N: associated with XLAG and Proud syndrome) predicted to affect the nuclear localisation sequences (NLS) flanking either end of the ARX homeodomain. The NLS regions are required for correct nuclear import facilitated by Importin 13 (IPO13). We demonstrate that missense mutations in either the N- or C-terminal NLS regions of the homeodomain cause significant disruption to nuclear localisation of the ARX protein in vitro. Surprisingly, none of these mutations abolished the binding of ARX to IPO13. This was confirmed by co-immunoprecipitation and immmuno fluorescence studies. Instead, tagged and endogenous IPO13 remained bound to the mutant ARX proteins, even in the RanGTP rich nuclear environment. We also identify the microtubule protein TUBA1A as a novel interacting protein for ARX and show cells expressing mutant ARX protein accumulate in mitosis, indicating normal cell division may be disrupted.
We show that the most likely, common pathogenic mechanism of the missense mutations in NLS regions of the ARX homeodomain is inadequate accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13.
Mutations in the Aristaless-related homeobox gene (ARX) are associated with a wide variety of neurologic disorders including lissencephaly, hydrocephaly, West syndrome, Partington syndrome, and X-linked intellectual disability with or without epilepsy. A genotype-phenotype correlation exists for ARX mutations, however the molecular basis for this association has not been investigated. To begin understanding the molecular basis for ARX mutations, we tested the DNA binding sequence preference and transcriptional repression activity for Arx, deletion mutants and mutants associated with various neurologic disorders. We found DNA binding preferences of Arx are influenced by the amino acid sequences adjacent to the homeodomain. Mutations in the homeodomain show a loss DNA binding activity, while the T333N and P353R homeodomain mutants still possess DNA binding activities, although less than wild type. Transcription repression activity, the primary function of ARX, is reduced in all mutants except the L343Q, which has no DNA binding activity and does not functionally repress Arx targets. These data indicate that mutations in the homeodomain result in not only a loss of DNA binding activity but also loss of transcriptional repression activity. Our results provide novel insights into the pathogenesis of ARX related disorders and possible directions to pursue potential therapeutic interventions.
ARX; lissencephaly; X-linked intellectual disability and Homeodomain
Infantile spasms syndrome (ISS) is a catastrophic pediatric epilepsy with motor spasms, persistent seizures, mental retardation, and in some cases, autism. One of its monogenic causes is an insertion mutation (c.304ins (GCG)7) on the X chromosome, expanding the first polyalanine tract of the interneuron-specific transcription factor ARX from 16 to 23 alanine codons. Null mutation of the Arx gene impairs GABA- and cholinergic interneuronal migration but results in a neonatal lethal phenotype. We developed the first viable genetic mouse model of ISS that spontaneously recapitulates salient phenotypic features of the human triplet-repeat expansion mutation. Arx (GCG)10+7 (“Arx Plus7”) pups display abnormal spasm-like myoclonus and other key EEG features, including multifocal spikes, electrodecremental episodes, and spontaneous seizures persisting into maturity. The neurobehavioral profile of Arx mutants was remarkable for lowered anxiety, impaired associative learning, and abnormal social interaction. Laminar decreases of Arx+ cortical interneurons and a selective reduction of calbindin-, but not parvalbumin- or calretinin-expressing interneurons in neocortical layers and hippocampus indicate that specific classes of synaptic inhibition are missing from the adult forebrain, providing a basis for the seizures and cognitive disorder. A significant reduction of calbindin, NPY-expressing and cholinergic interneurons in the mutant striatum suggest that dysinhibition within this network may contribute to the dyskinetic motor spasms. This mouse model narrows the range of critical pathogenic elements within brain inhibitory networks essential to recreate this complex neurodevelopmental syndrome.
migration; interneuron; GCG repeat mutation; epilepsy; autism; transcription factor
Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic α cell fate specification while simultaneously repressing the β and δ cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the α cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of β and δ cells. Concurrently, a remarkable increase in the number of cells displaying α cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult β cells led to a loss of the β cell phenotype and a concomitant increase in a number of cells with α or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of β cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.
Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc 12a5, Ets2, Phlda1, Egr1, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, and Napb) and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b, and Slit2) in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy.
ARX; GABA; epilepsy; interneurons; neuronal migration; basal ganglia
Defects in glucagon action can cause hyperplasia of islet α-cells, however, the underlying mechanisms remain largely to be elucidated. Mice homozygous for a glucagon-GFP knock-in allele (Gcggfp/gfp) completely lack proglucagon-derived peptides and exhibit hyperplasia of GFP-positive α-like cells. Expression of the transcription factor, aristaless-related homeobox (ARX), is also increased in the Gcggfp/gfp pancreas. Here, we sought to elucidate the role of ARX in the hyperplasia of α-like cells through analyses of two Arx mutant alleles (ArxP355L/Y and Arx [330insGCG]7/Y) that have different levels of impairment of their function. Expression of Gfp and Arx genes was higher and the size and number of islets increased in the Gcggfp/gfp pancreas compared to and Gcggfp/+ pancreas at 2 weeks of age. In male Gcggfp/gfp mice that are hemizygous for the ArxP355L/Y mutation that results in a protein with a P355L amino acid substitution, expression of Gfp mRNA in the pancreas was comparable to that in control Gcggfp/+Arx+/Y mice. The increases in islet size and number were also reduced in these mice. Immunohistochemical analysis showed that the number of GFP-positive cells was comparable in Gcggfp/gfp ArxP355L/Y and Gcggfp/+Arx+/Y mice. These results indicate that the hyperplasia is reduced by introduction of an Arx mutation. ArxP355L/Y mice appeared to be phenotypically normal; however, Arx [330insGCG]7/Y mice that have a mutant ARX protein with expansion of the polyalanine tract had a reduced body size and shortened life span. The number of GFP positive cells was further reduced in the Gcggfp/gfp Arx [330insGCG]7/Y mice. Taken together, our findings show that the function of ARX is one of the key modifiers for hyperplasia of islet α-like cells in the absence of proglucagon-derived peptides.
Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations.
The Arx transcription factor is expressed in the developing ventral telencephalon and subsets of its derivatives. Mutation of human ARX ortholog causes neurological disorders including epilepsy, lissencephaly, and mental retardation. We have isolated the mouse Arx endogenous enhancer modules that control its tightly compartmentalized forebrain expression. Interestingly, they are scattered downstream of its coding region and partially included within the introns of the downstream PolA1 gene. These enhancers are ultraconserved noncoding sequences that are highly conserved throughout the vertebrate phylum. Functional characterization of the Arx GABAergic enhancer element revealed its strict dependence on the activity of Dlx transcription factors. Dlx overexpression induces ectopic expression of endogenous Arx and its isolated enhancer, whereas loss of Dlx expression results in reduced Arx expression, suggesting that Arx is a key mediator of Dlx function. To further elucidate the mechanisms involved, a combination of gain-of-function studies in mutant Arx or Dlx tissues was pursued. This analysis provided evidence that, although Arx is necessary for the Dlx-dependent promotion of interneuron migration, it is not required for the GABAergic cell fate commitment mediated by Dlx factors. Although Arx has additional functions independent of the Dlx pathway, we have established a direct genetic relationship that controls critical steps in the development of telencephalic GABAergic neurons. These findings contribute elucidating the genetic hierarchy that likely underlies the etiology of a variety of human neurodevelopmental disorders.
basal forebrain; development; epilepsy; GABAergic neuron; neuronal progenitor cell; basal ganglia
Aristaless-related homeobox (ARX) gene mutations cause a diverse spectrum of disorders of the human brain, including lissencephaly, various forms of epilepsy and non-syndromic mental retardation. We have identified a novel mutation, c.81C>G (p.Y27X), within the ARX gene in a family with two affected male cousins. One of the boys was diagnosed with an early infantile epileptic encephalopathy also known as Ohtahara syndrome, whereas his cousin had been diagnosed with West syndrome (WS). Both patients have normal genitalia and neither have lissencephaly. The ARX mutation identified is predicted to yield a severely truncated protein of only 26 amino acids and can be considered as a null mutation. Somewhat surprisingly, however, it does not yield the X-linked lissencephaly with ambiguous genitalia (XLAG) syndrome. We proposed that the ARX mRNA translation re-initiated at the next AUG codon at position c.121–123 (aa 41) and, thus, partly rescued these patients from XLAG. Our in vitro studies show that this N-terminally truncated ARX protein (p.M41_C562) is detected by western immunoblot in lysates from cells transiently transfected with an ARX over-expression construct containing the c.81C>G mutation. Although these findings widen the spectrum of clinical phenotypes because of mutations in the ARX gene, they also emphasize the molecular pathogenetic effect of individual mutations as well as the effect of genetic background resulting in intrafamilial clinical heterogeneity for these mutations.
Ohtahara syndrome; burst suppression; ARX gene; West syndrome
ARX/Arx is a homeodomain-containing transcription factor necessary for the specification and early maintenance of pancreatic endocrine α-cells. Many transcription factors important to pancreas development, including ARX/Arx, are also crucial for proper brain development. Although null mutations of ARX in human patients result in the severe neurologic syndrome XLAG (X-linked lissencephaly associated with abnormal genitalia), the most common mutation is the expansion of the first polyalanine tract of ARX, which results primarily in the clinical syndrome ISSX (infantile spasms). Mouse models of XLAG, ISSX and other human ARX mutations demonstrate a direct genotype-phenotype correlation in ARX-related neurologic disorders. Furthermore, mouse models utilizing a polyalanine tract expansion mutation have illustrated critical developmental differences between null mutations and expansion mutations in the brain, revealing context-specific defects. Although Arx is known to be required for the specification and early maintenance of pancreatic glucagon-producing α-cells, the consequences of the Arx polyalanine expansion on pancreas development remain unknown. Here we report that mice with an expansion mutation in the first polyalanine tract of Arx exhibit impaired α-cell specification and maintenance, with gradual α-cell loss due to apoptosis. This is in contrast to the re-specification of α-cells into β- and δ-cells that occurs in mice null for Arx. Overall, our analysis of an Arx polyalanine expansion mutation on pancreatic development suggests that impaired α-cell function might also occur in ISSX patients.
The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, Aristaless-related homeobox gene (Arx) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of Arx in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of Arx, we have generated mice in which the Arx gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of Arx in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these Arx-deficient converted cells express β-cell markers including Pdx1, MafA, and Glut2. Surprisingly, short-term ablation of Arx in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for Arx in maintaining α-cell identity.
Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect.
We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing.
MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified.
Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.
Intellectual disability is common. Aristaless-related homeobox (ARX) gene is one of the most frequently mutated and pleiotropic genes, implicated in 10 different phenotypes. More than half of ∼100 reported cases with ARX mutations are due to a recurrent duplication of 24 bp, c.429_452dup, which leads to polyalanine tract expansion. The excess of affected males among the offspring of the obligate carrier females raised the possibility of transmission ratio distortion for the c.429_452dup mutation. We found a significant deviation from the expected Mendelian 1:1 ratio of transmission in favour of the c.429_452dup ARX mutation. We hypothesise that the preferential transmission of the c.429_452dup mutation may be due to asymmetry of meiosis in the oocyte. Our findings may have implications for genetic counselling of families segregating the c.429_452dup mutation and allude to putative role of ARX in oocyte biology.
intellectual disability; polyalanine tract expansions; Mendelian transmission; ARX; meiotic drive
Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations.
Arx; transcription factor; glucagon; somatostatin; gastrin; CCK; secretin; serotonin; enteroendocrine cells; endocrine progenitors; specification; differentiation
Genetic investigations of X-linked mental retardation have demonstrated the implication of ARX in a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities, but with associated features of dystonia and epilepsy. These investigations have in recent years directed attention to the role of this gene in brain development. Analysis of its spatio-temporal localization profile revealed expression in telencephalic structures at all stages of development, mainly restricted to populations of GABA-containing neurons. Furthermore, studies of the effects of ARX loss of function either in humans or in lines of mutant mice revealed varying defects, suggesting multiple roles of this gene during development. In particular, Arx has been shown to contribute to almost all fundamental processes of brain development: patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. In this review, we will present and discuss recent findings concerning the role of ARX in brain development and how this information will be useful to better understand the pathophysiological mechanisms of mental retardation and epilepsy associated with ARX mutations.
ARX; GABA; lissencephaly; epilepsy; interneurons; neuronal migration; basal ganglia
Mice lacking the Dlx1 and Dlx2 homeobox genes (Dlx1/2 mutants) have severe deficits in subpallial differentiation, including overexpression of the Gsx1 and Gsx2 homeobox genes. To investigate whether Gsx overexpression contributes to the Dlx1/2 mutant phenotypes, we made compound loss-of-function mutants. Eliminating Gsx2 function from the Dlx1/2 mutants rescued the increased expression of Ascl1 and Hes5 (Notch signaling mediators) and Olig2 (oligodendrogenesis mediator). In addition, Dlx1/2;Gsx2 mutants, like Dlx1/2;Ascl1 mutants, exacerbated the Gsx2 and Dlx1/2 patterning and differentiation phenotypes, particularly in the lateral ganglionic eminence (LGE) caudal ganglionic eminence (CGE), and septum, including loss of GAD1 expression. On the other hand, eliminating Gsx1 function from the Dlx1/2 mutants (Dlx1/2;Gsx1 mutants) did not severely exacerbate their phenotype; on the contrary, it resulted in a partial rescue of medial ganglionic eminence (MGE) properties, including interneuron migration to the cortex. Thus, despite their redundant properties, Gsx1 and -2 have distinct interactions with Dlx1 and -2. Gsx2 interaction is strongest in the LGE, CGE, and septum, whereas the Gsx1 interaction is strongest in the MGE. From these studies, and earlier studies, we present a model of the transcriptional network that regulates early steps of subcortical development. J. Comp. Neurol. 521:1561–1584, 2013. © 2012 Wiley Periodicals, Inc.
Dlx; Gsx; CGE; LGE; MGE; septum; development; mutant; interneuron; GABA; basal ganglia
Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). Herein we demonstrate that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform towards a subtype of GABAeric striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome).
The homeobox-containing aristaless-related protein ARX has been directly linked to the development of a number of human disorders involving mental retardation and epilepsy and clearly plays a critical role in development of the vertebrate central nervous system. In this work, we investigate the role of ALR-1, the Caenorhabditis elegans aristaless orthologue, in amphid sensory function. Our studies indicate that ALR-1 is required for maintenance of the amphid organ structure throughout larval development. Mutant analysis indicates a progressive loss in the amphid neurons' ability to fill with lipophilic dyes as well as a declining chemotactic response. The degeneration in amphid function corresponds with a failure of the glial-like amphid socket cell to maintain its specific cell shape and cell–cell contacts. Consistent with ALR-1 expression within the amphid socket cell, our results indicate a cell autonomous role for ALR-1 in maintaining cell shape. Furthermore, we demonstrate a role for ALR-1 in the proper morphogenesis of the anterior hypodermis. Genetic interaction tests also suggest that ALR-1 may function cooperatively with the cell adhesion processes in maintaining the amphid sensory organs.
A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation.
Dlx5 and Dlx6 homeobox genes are expressed in developing and mature cortical interneurons. Simultaneous deletion of Dlx5 and 6 results in exencephaly of the anterior brain; despite this defect, prenatal basal ganglia differentiation appeared largely intact, while tangential migration of Lhx6+ and Mafb+ interneurons to the cortex was reduced and disordered. The migration deficits were associated with reduced CXCR4 expression. Transplantation of mutant immature interneurons into a wild type brain demonstrated that loss of either Dlx5, or Dlx5&6, preferentially reduced the number of mature parvalbumin+ interneurons; those parvalbumin+ interneurons that were present had increased dendritic branching es. Dlx5/6+/- mice, which appear normal histologically, show spontaneous electrographic seizures, and reduced power of gamma oscillations. Thus, Dlx5&6 appeared to be required for development and function of somal innervating (parvalbumin+) neocortical interneurons. This contrasts with Dlx1, whose function is required for dendrite innervating (calretinin+, somatostatin+ and neuropeptide Y+) interneurons (Cobos et al., 2005).
Dlx5; Dlx6; Cortical Interneuron; Parvalbumin; Transplantation; Subtype Specification
Infantile spasms is a developmental epilepsy syndrome with unique clinical and EEG features, a specific pattern of pharmacological responsiveness, and poor outcome in terms of cognition and epilepsy. Despite the devastating nature of infantile spasms, little is known about its pathogenesis. Until recently, there has been no animal model available to investigate the pathophysiology of the syndrome or to generate and test novel therapies. Now, several promising animal models have emerged, spanning the etiological spectrum from genetic causes (e.g., Down syndrome or Aristaless-related homeobox [ARX] mutation) to acquired causes (e.g., endogenous and exogenous toxins or stress hormones with convulsant activity or blockade of neural activity). These new models are discussed in this review, with emphasis on the insights each can provide for understanding, treating, and preventing infantile spasms.
DLX1 and DLX2 transcription factors are necessary for forebrain GABAergic neuron differentiation, migration, and survival. We generated transgenic mice that express Cre-recombinase under the control of two ultra-conserved DNA elements near the Dlx1&2 locus termed I12b and URE2. We show that Cre-recombinase is active in a “Dlx-pattern” in the embryonic forebrain of transgenic mice. I12b-Cre is more active than URE2-Cre in the medial ganglionic eminences and its derivatives. Fate-mapping of EGFP+ cells in adult Cre;Z/EG animals demonstrated that GABAergic neurons, but not glia, are labeled. Most NPY+, nNOS+, parvalbumin+, and somatostatin+ cells are marked by I12b-Cre in the cortex and hippocampus, while 25-40% of these interneuron subtypes are labeled by URE2-Cre. Labeling of neurons generated between E12.5 to E15.5 indicated differences in birth-dates of EGFP+ cells that populate the olfactory bulb, hippocampus, and cortex. Finally, we provide the first in vivo evidence that both I12b and URE2 are direct targets of DLX2 and require Dlx1 and Dlx2 expression for proper activity.
X-linked mental retardation (XLMR) is the leading cause of mental retardation in males. Mutations in the ARX gene in Xp22.1 have been found in numerous families with both nonsyndromic and syndromic XLMR. The most frequent mutation in this gene is a 24 bp duplication in exon 2. Based on this fact, a panel of XLMR families linked to Xp22 was tested for this particular ARX mutation.
Genomic DNA from XLMR families linked to Xp22.1 was amplified for exon 2 in ARX using a Cy5 labeled primer pair. The resulting amplicons were sized using the ALFexpress automated sequencer.
A panel of 11 families with X-linked mental retardation was screened for the ARX 24dup mutation. Four nonsyndromic XLMR families – MRX29, MRX32, MRX33 and MRX38 – were found to have this particular gene mutation.
We have identified 4 additional XLMR families with the ARX dup24 mutation from a panel of 11 XLMR families linked to Xp22.1. This finding makes the ARX dup24 mutation the most common mutation in nonsyndromic XLMR families linked to Xp22.1. As this mutation can be readily tested for using an automated sequencer, screening should be considered for any male with nonsyndromic MR of unknown etiology.