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
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.
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.
Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6CRE-IRES-GFP(Dlx5/6CIG) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx−/y;Dlx5/6CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx−/+;Dlx5/6CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.
Epilepsy; development; conditional knockout; genetic model; interneurons
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.
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
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
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.
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) 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.
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.
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (ArxE), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although ArxE functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of ArxE to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
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.
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
Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.
Nkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2null;ArxΔpanc) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2null;ArxΔpanc mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells.
Nkx2.2; Arx; transcriptional regulation; endocrine cell fate; ghrelin; PP; somatostatin
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
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
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.
Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3+ endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3+) and in early differentiating (ChromograninA−) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.
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
Adult pancreatic beta cells can replicate during growth and after injury to maintain glucose homeostasis. Here we report that beta cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to alpha cells. We identified the lineage determination gene aristaless related homeobox (Arx), as methylated and repressed in beta cells, and hypo-methylated and expressed in alpha cells and Dnmt1-deficient beta cells. We show that the methylated region of the Arx locus in beta cells was bound by methyl binding protein MeCP2 which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of alpha cell lineage determination genes to maintain pancreatic beta cell identity.
While many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described.
Here we show that mutations in genes encoding the Arp2/3 complex (arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length.
Our results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that, in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth.
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.
Retrotransposed sequences arise from messenger RNAs (mRNAs) that have been reinserted into genomic DNA by reverse transcription. Usually, these sequences are embedded in dormant regions, collect missense mutations over time and constitute processed, nonfunctional pseudogenes. There are thousands of processed pseudogenes in the mouse and human genome. Here, we report evidence for two paralog genes (termed Arxes1 and Arxes2), which arose by retrotransposition of the signal peptidase Spcs3 followed by a segmental duplication event. They gained a functional promoter that we show to be transactivated by adipogenic transcription factors. We further show that the Arxes mRNAs are highly expressed in adipose tissue and strongly upregulated during adipogenesis in different cell models. Additionally, their expression is elevated by an anti-diabetic agent in vitro and in vivo. Importantly, we provide evidence that the Arxes genes are translated and that the proteins are located in the endoplasmic reticulum. Although the sequence similarity and subcellular location are reminiscent of their parental gene, our data suggest that the Arxes have developed a different function, since their expression is required for adipogenesis, whereas Spcs3 is dispensable. In summary, we report retrotransposed-duplicated genes that evolved from a parental gene to function in a tissue and adipogenesis-specific context.