Interactive Fly, Drosophila

aristaless


EVOLUTIONARY HOMOLOGS (part 1/2)

Invertebrate Aristalless-related proteins

Two homeobox genes, prdl-a and prdl-b, which were isolated from a Hydra vulgaris cDNA library, encode paired-like class homeodomains highly related to those of the aristaless-related genes. In adult polyps, prdl-b is a marker for synchronously dividing nematoblasts while prdl-a displays an expression restricted to the the nerve cell lineage of the head region. During budding and apical regeneration, an early and transient prdl-a expression is observed in endodermal cells of the stump at a time when the head organizer is established. When apical regeneration is delayed upon concomittant budding, prdl-a expression is found to be altered in the stump. A specific anti-prdl-a protein immunoserum reveals that prdl-a is overexpressed in adult polyps of the Chlorohydra viridissima multiheaded mutant, with an expression domain extending below the tentacle ring towards the body column. Accordingly, prdl-a DNA-binding activity is enhanced in nuclear extracts from this mutant. These results suggest that prdl-a responds to apical forming signals and might thus be involved in apical specification. When a marine hydrozoan (Podocorynae carnea) is examined, the anti-prdl-a antibody shows cross-reactivity with cells located around the oral region, indicating that prdl-a function is shared by other cnidaria. The ancestral role for prdl-a-related genes in the molecular definition of the head (or oral-surrounding region) is discussed (Gauchat, 1998).

In cnidaria, the region surrounding the mouth opening is involved in food detection and ingestion, and thus named the head. In hydra, nerve cell density is maximal in the head region with, in some species, a nerve ring at the base of the tentacle insertion ring. Contraction-burst potentials, a process that is altered upon light exposure, originate in the hypostome and are conducted throughout the body column. Thus, the head is the place where a high level of cellular and morphological organisation correlates with complex behaviours. In triploblastic species, food detection, ingestion and partial processing are also located in the head region, which contains sense organs, complex neural structures and, in vertebrates, the distribution of respiratory gases. Most of the vertebrate head has supposedly arisen de novo, rather than by modification of a preexisting structure. However, whether ancestral elements defining a 'minimal head region' might be present in less complex species, including diploblastic species, remains an open question. In vertebrates, regulatory genes have been isolated that are parts of a molecular head-organizing activity. For example, twist, a basic helix-loop-helix transcription factor, regulates differentiation and behaviour of head mesenchymal cells in mice. The Lim-1 gene, a LIM-class homeobox gene, is required for formation of an early organized node and anterior axial mesoderm, whereas the Cart1 gene is required for the proliferation of forebrain mesenchyme cells: Cart1-deficient mice display strong anomalies of neural tube closure consecutive to head mesenchyme defects. Cart-1 is a cartilage specific paired type homeodomain protein. The early and transient pattern of prdl-a expression during regeneration suggests a function for prdl-a in the differentiation of the most apical part of the animal. It is thus tempting to speculate that this morphogenetic role is reminiscent of that observed and/or supposed for Arx in vertebrate brain development and Cart1 in mouse head formation (Gauchat, 1998).

Developmental gradients are known to play important roles in axial patterning in hydra. Current efforts are directed toward elucidating the molecular basis of these gradients. HyAlx, an aristaless-related gene in hydra has been isolated and characterized. The expression patterns of the gene in adult hydra, as well as during bud formation, head regeneration and the formation of ectopic head structures along the body column, indicate the gene plays a role in the specification of tissue for tentacle formation. The use of RNAi provides more direct evidence for this conclusion. The different patterns of HyAlx expression during head regeneration and bud formation also provide support for a recent version of a reaction-diffusion model for axial patterning in hydra (Smith, 2000).

The de novo formation of tentacles occurs during budding, head regeneration and as a result of DAG treatment. Although the initial stages of HyAlx expression vary in the three developmental contexts, the latter stages are the same. Immediately prior to tentacle formation, a necklace of spots of intense HyAlx expression appear. These spots transform into rings as HyAlx expression vanishes from the center of each spot, and subsequently, tentacles emerge from the centers of the rings. In addition, the HyAlx pattern during the formation of DAG-induced tentacles demonstrates that this phase of HyAlx expression is not related to a general head-patterning process, but is specifically related to the formation of tentacles. Two additional observations illustrate that HyAlx is a very precise marker for the tissue which is going to form a tentacle. (1) There appears to be a correlation between HyAlx spot size and tentacle size in both budding and regeneration: large diameter spots give rise to large diameter tentacles, while smaller spots give rise to tentacles with smaller diameters. (2) The appearance of HyAlx spots is tightly coupled with the timing of the initial evagination of tentacles. The spots appear sequentially in the same order as the order of the emergence of individual tentacles during budding. The first two spots appear on the basal side of the bud, which is also where the first two tentacles arise on these buds. Thus, the spot and ring pattern of HyAlx expression is consistently associated with the emergence of tentacles, indicating the gene is involved in the specification of tissue to form tentacles. The RNAi experiments provide more direct evidence for this conclusion. After introducing HyAlx dsRNA into developing buds at a stage before HyAlx expression has begun, the appearance of tentacles is delayed significantly compared with controls. This delay is specifically due to the HyAlx dsRNA since a control dsRNA, namely luciferase dsRNA, had very little effect on the appearance of tentacles. The fact that the dsRNA treatment causes a delay, but does not eliminate tentacle formation, reflects the continuously regulative nature of hydra tissue. Since the patterning processes are continuously active, while the presence of dsRNA is transient, interference with a gene affecting tentacle formation would also be expected to be transient, but not permanent (Smith, 2000).

Once tentacles are formed, HyAlx continues to play a role in tentacle patterning in the adult. In the adult, as cells are displaced from the tentacle zone onto the tentacles, they undergo changes in their cellular properties. While in the tentacle zone, the epithelial cells are continually proliferating, but as they cross the tentacle zone/tentacle border and enter a tentacle, they become permanently arrested in the G2-phase of the cell cycle. At the same time, the ectodermal epithelial cells undergo terminal differentiation to form tentacle-specific battery cells. The border between tentacle zone and tentacle is sharp and very precise, so that a cell on the tentacle zone side of the border exhibits dramatically different properties from its immediate neighbor on the tentacle side. This abrupt transition is reflected in the expression of several molecular markers. CnOtx, an Otx gene and Cnox3, a Hox gene are expressed in the ectodermal epithelial cells of the tentacle zone. The expression of both of these genes stops suddenly at the border, so that neither Cnox3 nor CnOtx is expressed in the tentacle. Conversely, as cells cross the border, several genes not expressed in the tentacle zone are expressed at a high level as soon as these ectodermal cells enter the tentacle. These include an insulin receptor homolog, HTK; an annexin gene, TS19, which is a cell-surface antigen, and a hydra metalloproteinase, HMP1 (Smith, 2000).

HyAlx is expressed in rings of ectodermal cells that are approximately 3-4 cells wide, bridging this border. As ectodermal cells are displaced through the tentacle zone, they abruptly begin to express HyAlx, then cross the border, and only a couple of cell diameters past the border, they stop expressing HyAlx. Its expression at this border suggests that HyAlx might be involved in initiating some of the changes which take place in the tentacle zone cells as they prepare to cross the border. For example, HyAlx could have a role in driving cells from a proliferative to a differentiated state. The gene could also, or instead, be involved in changes in cell shape, since the ectodermal cells switch from columnar body column cells to the flat battery cells of the tentacle. In sum, HyAlx is very tightly associated with the patterning of tentacles. The gene appears to be involved in the specification of patches of cells in a developing head to form tentacles, as well as in the specification of tentacle zone tissue to become tentacle tissue in the context of continuous tissue movement in the adult (Smith, 2000).

Patterns of aristaless of Gryllus bimaculatus, a hemimetabola model insect, are reported. Gryllus aristaless (Gbal) is expressed in the most distal region of developing labrum, antenna, mandible, maxilla, labium, leg, cercus, and hindgut. Gbal is also expressed in the proximal region, corresponding to the presumptive coxopodite of the developing antenna, mandible, maxilla, labium, and leg, but not in the developing labrum, cercus, and hindgut. During development of the leg, expression of Gbal changes dynamically with the progress in leg segmentation: Gbal is expressed in order in the presumptive pretarsus, coxa, femur, tibia and tarsus before appearance of morphological segmentation. Morphological segmentation follows Gbal expression in a proximodistal order from ED2 to ED5 after expression in the most-distal region (ED1). The essential features of Gbal expression patterns, in the Gryllus leg bud at early stages, resemble those in the Drosophila leg imaginal disc (Miyawaki, 2002).

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. A new and essential component has been identified of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through ß-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. These findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein (Ettensohn, 2003).

Regulation of chemosensory and GABAergic motor neuron development by the C. elegans Aristaless/Arx homolog alr-1

Mutations in the highly conserved Aristaless-related homeodomain protein ARX have been shown to underlie multiple forms of X-linked mental retardation. Arx knockout mice exhibit thinner cerebral cortices because of decreased neural precursor proliferation, and also exhibit defects in the differentiation and migration of GABAergic interneurons. However, the role of ARX in the observed behavioral and developmental abnormalities is unclear. The regulatory functions of individual homeodomain proteins and the networks in which they act are frequently highly conserved across species, although these networks may be deployed in different developmental contexts. In Drosophila, aristaless mutants exhibit defects in the development of terminal appendages, and Aristaless has been shown to function with the LIM-homeodomain protein LIM1 to regulate leg development. This study describes the role of the Aristaless/Arx homolog alr-1 in C. elegans. alr-1 acts in a pathway with the LIM1 ortholog lin-11 to regulate the development of a subset of chemosensory neurons. Moreover, the differentiation of a GABAergic motoneuron subtype is affected in alr-1 mutants, suggesting parallels with ARX functions in vertebrates. Investigating ALR-1 functions in C. elegans may yield insights into the role of this important protein in neuronal development and the etiology of mental retardation (Melkman, 2005).

The results indicate that ALR-1 acts in distinct transcriptional cascades to regulate asymmetric cell division of a neuronal precursor and to specify the characteristics of a GABAergic MN subtype in C. elegans. These processes have parallels to the processes regulated by ARX in vertebrates. In arx mutant mice, neuroblast proliferation in the cerebral cortex is decreased. Neuroblast proliferation in the ventricular zone occurs via temporally regulated symmetric and asymmetric cell divisions that generate additional neuronal precursors and postmitotic neurons. It is speculated that ARX may regulate these cell divisions perhaps by regulating the localization or segregation of determinants such as Numb or Notch. ALR-1 acts in part by temporally restricting expression of lin-11 in the AWA neurons, and by promoting lin-11 expression in the ASG neurons. Interestingly, expression of the LIM homeobox genes Lhx6 and Lhx9 is abolished in the neocortex and thalamic eminence, respectively, in Arx mutant mice, whereas the domain of Lhx6 expression in the ganglionic eminences is enlarged. Taken together with the observation that lim1 and al function in a network to regulate Drosophila leg development, these findings suggest that regulatory mechanisms between ARX proteins and LIM-HD proteins may be conserved across species (Melkman, 2005).

ALR-1 acts together with the UNC-55 COUP transcription factor to regulate the differentiation of a GABAergic MN type in C. elegans. A COUP-TF protein and the PRDL-B Aristaless/ARX homolog have been shown to act in a network to regulate neurogenesis in Hydra. In vertebrates, COUP transcription factors have been implicated in neurogenesis, neuronal differentiation, migration and axonal guidance. Interestingly, COUP-TFI and COUP-TFII exhibit overlapping spatiotemporal expression patterns with ARX in the developing neocortex, as well as in the lateral and medial ganglionic eminences, which give rise to GABAergic interneurons. Moreover, COUP-TFI is co-expressed with the GABAergic neuron marker calbindin in the cortex. These findings suggest the intriguing possibility that COUP and ARX function together to regulate neuronal, and in particular GABAergic, neuronal development. These results suggest that ARX proteins function in partly conserved genetic networks to regulate the development of different tissue and cell types in different species, and raise the possibility that identification of potential interactors and targets of ALR-1 in C. elegans may aid in elucidating ARX function in brain development in vertebrates (Melkman, 2005).

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT (Saunders, 2014).

Anti-intron antisense RNA blocks embryonic alx1 gene expression

Many genes, and particularly regulatory genes, are utilized multiple times in unrelated phases of development. For studies of gene function during embryogenesis, there is often need of a method for interfering with expression only at a specific developmental time or place. In sea urchin embryos cis-regulatory control systems which operate only at specific times and places can be used to drive expression of short designed sequences targeting given primary transcripts, thereby effectively taking out the function of the target genes. The active sequences are designed to be complementary to intronic sequences of the primary transcript of the target genes. In this work, the target genes were the transcription factors alx1 and ets1, both required for skeletogenesis, and the regulatory drivers were from the sm30 and tbr genes. The sm30 gene is expressed only after skeletogenic cell ingression. When its regulatory apparatus was used as driver, the alx1 and ets1 repression constructs had the effect of preventing postgastrular skeletogenesis, while not interfering with earlier alx1 and ets1 function in promoting skeletogenic mesenchyme ingression. In contrast, repression constructs using the tbr driver, which is active in blastula stage, block ingression. This method thus provides the opportunity to study regulatory requirements of skeletogenesis after ingression, and may be similarly useful in many other developmental contexts (Smith, 2008).

The following experiment shows the potential effectiveness and specificity of anti-intron antisense RNA (aiRNA), transcribed from a cis-regulatory expression vector, for blocking target gene expression. Use was made of the fact that sea urchin eggs concatenate injected linear DNA, and whatever constructs are injected, stably incorporate these together into a blastomere chromosome, whereafter the exogenous concatenate replicates together with the host DNA. Thus a mixture of a marker construct, an aiRNA generating construct, and a target construct was injected. The marker consisted of tbr cis-regulatory DNA driving an RFP gene as a reporter (tbr > RFP). It will express only in skeletogenic cells and will identify those cells which contain the exogenous mix of constructs. The target construct consisted of an alx1 BAC, containing its own endogenous cis-regulatory information as well as the complete gene, into the 5' UTR of which a GFP coding sequence had been inserted by homologous recombination. The aiRNA generating construct (tbr > aiRNA) consisted of the same tbr cis-regulatory sequence as in the marker construct, but here used to drive expression of the antisense transcripts. In one version, the construct produced an antisense transcript targeting the intron1/exon1 splice junction of the alx1 gene [as would be targeted by a splice-blocking MASO (morpholino-substituted antisense oligonucleotides)]; and in a second version, it produced an antisense transcript targeting an internal region of intron1. These aiRNA transcripts were generated off 24 bp antisense oligonucleotides terminated with three tandem p(A) addition sites, cloned into the tbr expression vector. The results were monitored by QPCR measurement of GFP mRNA, normalized to the RFP mRNA in the same embryos. Both aiRNA constructs almost eliminated GFP mRNA production. Constructs generating sense rather than antisense transcripts of the same intronic sequences had no effect. Nor was expression of the alx1 BAC-GFP reporter affected by an aiRNA construct targeted against the ets1 gene, which is active in the same cells, excluding a nonspecific interference with expression. The effect of aiRNA constructs on endogenous alx1 transcripts could not be directly measurede in the same experiment by QPCR, due to the mosaic incorporation of the targeting vector, since about 3/4 of the skeletogenic cells lack the exogenous DNA, and produce normal levels of alx1 in the same embryos. In contrast, since the constructs are co-incorporated, as noted above, in the alx1 BAC-GFP experiment, the three- to four-fold reduction in GFP transcript is the actual gene knockdown effect in those cells carrying the aiRNA construct. It is concluded (1) that intranuclear stoichiometry does indeed appear to favor efficient target acquisition by endogenously produced antisense transcripts; (2) that the interference with GFP production was not a general effect of interference with splicing machinery; (3) that this interference operates on internal as well as junctional intronic sequences; and (4) that it causes destruction or inactivation of the whole target transcript since the target sequences are all downstream of the intact GFP sequence. In other words, it is likely that the primary transcript is targeted for degradation. Though it is expected that the p(A) sites would ultimately result in short aiRNA transcripts, it is not known whether the active inhibitory form is a longer readthrough pre-poly(A) RNA, or the terminated polyadenylated product (Smith, 2008).

If the effect of an aiRNA construct is indeed the functional inactivation of the target transcript so that it cannot be expressed, then introduction of tbr > aiRNA against alx1 should produce the same morphological effect on the skeletogenic cells carrying it as does injection into the egg of MASO against alx1, since the tbr cis-regulatory control system initiates expression very early in development. The alx1 MASO effect is the total prevention of ingression: alx1 regulates downstream differentiation genes required for this distinct function. The result of introducing tbr > aiRNA against alx1 is indeed that in 80%-90% of embryos bearing tbr > aiRNA targeted to alx1, no skeletogenic cells bearing the construct whatsoever emerge from the vegetal wall of the embryo, and in the remainder only a few do. The skeletogenic cells bearing the construct are marked by expression of tbr > GFP (i.e., the 'marker' in these experiments is a tbr > GFP construct as opposed to the tbr > RFP construct. Expression of ets1 is also required for ingression, as shown by its MASO phenotype, and again, this phenotype is seen as well with tbr > aiRNA directed against an ets1 intron. Quantitatively, both aiRNA constructs are extremely effective in arresting ingression (Smith, 2008).

The problem outlined above can now be approached: how to study late alx1 and ets1 function by knocking out expression in skeletogenic cells only after allowing these genes to function long enough to permit complete ingression. To this end, the cis-regulatory system of the sm30 gene was used; it is turned on only after ingression. Sm30 > aiRNA constructs targeted against the same intronic sequences of either alx1 or ets1 as in the tbr > aiRNA constructs were introduced, together with the tbr > GFP marker construct. Assuming that sm30 cis-regulatory control is sufficiently tight, the expectation is that there will be no expression of the aiRNAs prior to ingression and thus that neither construct will interfere with ingression. In fact, both sets of embryos displayed control levels of ingression. However, subsequent skeletogenesis was dramatically affected, though in a very specific way. In normal postgastrular embryos, the skeletogenic cells migrate about the inner walls of the blastocoel, and then read signals displayed by the ectoderm cells, which specify the bilateral, branched form of the skeletal spicules. In response, they arrange themselves in highly reproducible, ordered, linear arrays. The cells then fuse laterally and secrete the skeleton into extracellular cables by which they are connected to one another. But the cells bearing sm30 > aiRNA targeted to either alx1 or ets1 fail entirely to form these arrays, or to participate in secretion of organized spicule rods. The cells instead assume random positions on the inner wall of the blastocoel: thus they retain their motility, but it would appear that they have failed to respond to the spatial information presented on the blastocoel wall. That this information is being normally expressed in the same embryos can be seen by the presence of morphologically normal skeletal elements formed by cells not bearing the aiRNA constructs, i.e., not expressing GFP. Secondary skeletogenic cells were not observed up to 72 h post-fertilization. The basic biomineralization functions are also severely affected. Thus instead of all tbr > GFP cells producing biomineral as in controls, only about 3% of green cells in the aiRNA embryos are associated with rudimentary accumulations of biomineral, which can be detected in polarized light. In summary, the experiment shows that expression of alx1 and ets1 after ingression is required for alignment of the cells in response to ectodermal patterning information; whether these genes are needed for syncytial cable formation is moot since they never get in position to form linear cables. Both alx1 and ets1 are clearly required for completion of the skeletogenic program. These functions are consistent with the character of the gene regulatory network linkages set up by the time of ingression, which include, for both genes, inputs into signal receptors and into biomineralization differentiation genes. It is now clear that these regulatory linkages are set up to be utilized only after ingression, and that they and no doubt many others of similar nature are requisite for mature skeletogenic function (Smith, 2008).

This study has shown that, in sea urchin embryos, expression of RNA complementary to intronic sequence, under control of selected cis-regulatory modules, can be used to effect spatially and temporally targeted gene expression knockdown. The results of the ets1 and alx1 aiRNA experiments are exactly consistent with expectation from the model experiment that demonstrated aiRNA efficacy against the alx1 BAC GFP construct (Smith, 2008).

The mechanism by which these interference constructs work is not known. It is clearly distinct from that of classical RNAi since the latter causes destruction of target mRNAs in the cytoplasm, a process nucleated on the RNAi:3' trailer complex. Messenger RNA destruction mediated by RNAi is effected by cytoplasmic proteins, while in the current case the sequence targeted, i.e., the intron, exists only in the nucleus. Nor does the mechanism of interference with expression seem the same as that of splice-blocking morpholinos, even though this project began with the thought that because of favorable stoichiometry the function of splice blocking morpholinos could be duplicated by use of endogenously synthesized antisense RNAs. Splice blocking morpholinos leave unspliced primary transcript fragments to accumulate in the nucleus where they are easily detected. But, as pointed out above, the aiRNA vectors apparently cause the destruction of the whole the transcript since even portions upstream of the targeted intron disappear. The actual mechanism by which formation of a 24 bp intron-antisense duplex effects primary transcript destruction will be most interesting to determine (Smith, 2008).

In the meantime, at the very least, this advance opens the way to exploration of a plethora of interesting problems in the regulatory control of postgastrular development and morphogenesis in the sea urchin embryo. The result will be to extend gene regulatory network analysis to the later development of this model embryonic system. The effectiveness of the method may depend on the intra-nuclear stoichiometric ratios of transcripts made on exogenous constructs to endogenous pre-mRNAs. Thus, the generality of its application in other model organisms would need to be determined (Smith, 2008).

Fish Aristaless-related proteins

A zebrafish paired-type homeobox gene, Alx, is closely related to the murine Chx10 and the gold fish Vsx-I homeodomain proteins. Alx, named because of its homology to Drosophila Aristaless, has a paired domain and an overall 31% homology to Aristaless. The homology between Alx and Aristaless does not extend beyond the homeodomain. However, the degree of sequence similarity between the vertebrate and the nematode genes (C. elegans ceh-10) is striking, as is the homology between their expression domains. Thus the Drosophila gene might not be a true homolog of the worm or vertebrate genes. Alx is first expressed at about 12 h post-fertilization (hpf) when optic vesicles appear. Its expression is restricted to the early retinal neuroepithelium, whereas no signal can be detected in the optic placode. Later, Alx expression follows the differentiation of the neural retina. Inhibition experiments with antisense oligonucleotides result in specific eye malformations that are reminiscent of the phenotype of ocular retardation (or) mice, caused by a spontaneous Chx10 mutation. The expression of other developmentally relevant genes such as pax(zf-a), pax(zf-b) and krx-20 is not affected in the antisense treated embryos. Alx is a possible target of pax(zf-a) because pax(zf-a) is turned on about 2 hours before Alx and their expression domains partially overlap (Barabino, 1997).

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers for developing structures that may facilitate the detection of discrete structural alterations. In this context, Ol-Prx 3 (Oryzias latipes-Prx 3) has been characterized. This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. The aristalless gene of Drosophila codes for a protein with nearly identical homeobox, but Al has a primary structure highly divergent outside the homeobox and nonhomologous expression domains. Sequence analysis reveals that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12, which with the human SHOX gene, is thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. The question of the evolutionary conservation of OG-12 genes outside the vertebrate phylum remains open. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. This paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish (Joly, 1997).

Xenopus Aristaless-related proteins

A novel Xenopus homeobox gene, Xenopus retinal homeobox 1 or Xrx1, belongs to the paired-like class of homeobox genes. Paired-like class genes have no paired-box domain and share other sequence characteristics. The homeodomain of Xrx1 is 71% homologous to Drosophila aristaless, 70% homologous to murine Chx10 and 66% homologous to C. elegans ceh-10. Although these genes could have been ancestrally related, they have certainly diverged functionally and structurally during evolution. Xrx1 is expressed in the anterior neural plate, and subsequently in the neural structures of the developing eye (neural retina and pigmented epithelium), and in other forebrain structures deriving from the anterior neural plate; these include the pineal gland (throughout development), the diencephalon floor and the hypophysis. Its rostral limit of expression corresponds to the chiasmatic ridge, which some authors consider as the anteriormost limit of the neural tube: thus, Xrx1 may represent one of the most anteriorly expressed homeobox genes reported to date. Moreover, its expression in organs implicated in the establishment of circadian rhythms, may suggest for Xrx1 a role in the genetic control of this function. Analysis of Xrx1 expression in embryos subjected to various treatments, or microinjected with different dorsalizing agents (noggin, Xwnt-8), suggests that vertical inductive signals leading to head morphogenesis are required to activate Xrx1 (Casarosa, 1997).

Mammalian Aristaless-related proteins

A novel paired homeodomain protein, PHD1, most closely related to C. elegans unc-4, has been identified by a differential RT-PCR method. Unc-4 has no paired domain and is thus grouped separately from paired-homeodomains into a prd-like class. PHD1 is expressed in a narrow layer adjacent to the ventricular zone of the dorsal spinal cord, immediately following expression of MASH1 (see Drosophila Achaete) but preceding overt neuronal differentiation. Some cells coexpressing MASH1 and PHD1 can be seen, suggesting that these two genes are sequentially activated within the same lineage. In the olfactory sensory epithelium, PHD1 expression not only follows but is dependent upon MASH1 function, suggesting that PHD1 acts downstream of MASH1. A sequential action of bHLH and paired homeodomain proteins is apparent in other neurogenic lineages and may be a general feature of both vertebrate and invertebate neurogenesis (Saito, 1996).

A novel homeobox gene (Arx) expressed in the mouse central nervous system has been isolated that shows striking similarity to the homeodomain of Drosophila al gene (85% identity) and in a 17 amino acid-sequence near the carboxyl-terminus. The C-peptide domain is found in several homeoproteins belonging to the paired-like class. The possible relation of the C-peptide domain with a conserved sequence in the Orthopedia homeoprotein is suggested by sequence similarity and the nearly identical positions of this sequence in the two proteins. Transactivation activity is reduced when this sequence is deleted from the Otp homoeprotein. The designation Arx (aristaless related homeobox gene) is given in consideration of its structural similarity to the al gene. Arx is highly conserved between mouse and zebrafish. Neuromeric expression in the forebrain and longitudinal expression in the floor plate are observed in mouse and zebrafish. The expression of Arx in the ganglionic eminence and ventral thalamus overlaps regionally with that of Dlx1, but the cell layer where Arx is expressed differs from that of the Dlx1. This gene is also expressed in the dorsal telencephalon (presumptive cerebral cortex) of mouse embryos. The structure and expression pattern of Arx with respect to any possible relationship to al and Dlx1 is discussed as well as the function of Arx in the floor plate. It is unlikely that Arx regulates Sonic Hedgehog in the floor plate since Arx is expressed later than Shh. Undue emphasis should not be placed on colocalization of Arx and the Dlx gene family expression in the forebrain, since expression of Arx and Dlx1 is found to differ in other regions (Miura, 1997)

A murine homeobox containing gene, Uncx4.1 has been characterized. The homeodomain sequence exhibits 88% identity to the C. elegans unc-4 protein at the amino acid level, 70% related to the aristal-less homeodomain, and 63% to 70% identical to some other paired-type related homeodomains. The protein is not considered an Aristal-less homolog. In situ hybridization analysis reveals that Uncx4.1 is expressed in the paraxial mesoderm, in the developing kidney, and the central nervous system. The most intriguing expression domain is the somite, where it is confined to the caudal part of the newly formed somite and subsequently restricted to the caudal domain of the developing sclerotome. In the central nervous system, Uncx4.1 is detected in the developing spinal cord, hindbrain, mesencephalon, and telencephalon. The temporal and spatial expression pattern suggests that Uncx4.1 may play an important role in kidney development and in the differentiation of the sclerotome and the nervous system (Mansouri, 1997).

Deletion of the SHOX region on the human sex chromosomes has been shown to result in idiopathic short stature, and has been proposed to play a role in the short stature associated with Turner syndrome. A human paired-related homeobox gene, SHOT, has been identified by virtue of its homology to the human SHOX and mouse OG-12 genes. SHOTa and SHOTb encode proteins with a homeodomain identical to murine OG-12 and human SHOX. This homeodomain shows the highest homology to the homeodomains of paired-related proteins, including Arx, Prx2/S8, Phox2, Drosophila aristaless, Pax-3, and Drg11. In addition to the homeodomain itself, several potential phosphorylation sites, a putative SH3 binding domain and a 14-amino acid residue motif at the C-terminal end ("OAR-domain") are highly conserved among the OG-12, SHOX, and SHOT proteins. The predicted SHOT and OG-12 proteins are 99% identical over their entire length while the overall homology between SHOT and SHOX amounts to only 83% at the amino acid level, demonstrating that the human SHOT is related more closely to the murine OG-12 than to SHOX. Two different isoforms were isolated, SHOTa and SHOTb, that have identical homeodomains and share a C-terminal 14-amino acid residue motif characteristic for craniofacially expressed homeodomain proteins. Differences between SHOTa and b reside within the N termini and an alternatively spliced exon in the C termini. In situ hybridization of the mouse equivalent, OG-12, on sections from staged mouse embryos detects highly restricted transcripts in the developing sinus venosus (aorta), female genitalia, diencephalon, mes- and myel-encephalon, nasal capsula, palate, eyelid, and in the limbs. SHOT maps to human chromosome 3q25-q26 and OG-12 maps within a syntenic region of the mouse on chromosome 3. Based on the localization and expression pattern of its mouse homolog during embryonic development, SHOT represents a candidate for the Cornelia de Lange syndrome. This syndrome was first described in 1933 and is characterized by growth and mental retardation (microcephaly), distinctive facial deformities including cleft palate, abnormally situated eyelids, and nose and ear deformities, as well as heart defects and reductive limb development. Interestingly, the expression of the mouse SHOT homologue, OG-12, is in perfect agreement with the features seen in Cornelia de Lange syndrome. It shows high expression levels in craniofacial tissues including the palate, nasal capsula, eyelid, and ear, as well as in heart (aorta), brain, and developing limbs (Blaschke, 1998).

The specification of noradrenergic neurotransmitter identity in neural crest stem cells (NCSCs) has been investigated. Retroviral expression of both wild-type and dominant-negative forms of the paired homeodomain transcription factor Phox2a, related to Drosophila Aristalless, indicates a crucial and direct role for this protein (and/or the closely related Phox2b) in the regulation of endogenous tyrosine hydroxylase (TH) and dopamine-beta hydroxylase (DBH) gene expression in these cells. In collaboration with cAMP, Phox2a can induce expression of TH but not of DBH or of panneuronal genes. Phox2 proteins are, moreover, necessary for the induction of both TH and DBH by bone morphogenetic protein 2 (BMP2) (which induces Phox2a/b) and forskolin. Phox2 proteins are also necessary for neuronal differentiation. These data suggest that Phox2a/b coordinates the specification of neurotransmitter identity and neuronal fate by cooperating with environmental signals in sympathetic neuroblasts (Lo, 1999).

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, their expression was examined in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations (Patzke, 2001).

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain but lack a paired domain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, the functional and genetic relationships between Alx4 and Cart1 have been analyzed. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions: (1) mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage; (2) there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures, and (3) double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development (Qu, 1999).

Aristaless-related genes encode a structurally defined group of homeoproteins that share a C-terminal stretch of amino acids known as the OAR- or aristaless domain. Many aristaless-related genes have been linked to major developmental functions, but the function of the aristaless domain itself is poorly understood. Expression and functional studies have shown that a subgroup of these genes, including Prx1, Prx2, Alx3, Alx4 and Cart1, is essential for correct morphogenesis of the limbs and cranium. The function of the aristaless domain has been demonstrated in vivo by ectopically expressing normal and mutated forms of Cart1 and Alx3. Ectopic expression of Cart1 in transgenic mice does not disturb development, whereas expression of a Cart1 form from which the aristaless domain has been deleted results in severe cranial and vertebral malformations. The Alx3 protein contains a divergent aristaless domain that appears not to be functional, since ectopic expression of Alx3 results in an altered phenotype irrespective of the presence of this aristaless domain. Linking the Cart1 aristaless domain to Alx3 extinguishes teratogenicity. At the molecular level, the most important consequence of deleting the aristaless domain is increased DNA binding to its palindromic target sequence. This demonstrates that the aristaless domain functions as an intra-molecular switch to contain the activity of the transcription factor of which it is a part (Brouwer, 2003).

Mutation of Aristaless-related proteins

A paired-like homeodomain protein called Alx-4 has been isolated. Mice homozygous for a targeted null mutation of Alx-4 have several abnormalities, including preaxial polydactyly, suggesting that Alx-4 plays a role in pattern formation in limb buds. Alx-4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial-mesenchymal interactions, many of which are additionally dependent on expression of the HMG-box-containing protein, LEF-1. Alx-4-expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. Alx-4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N-terminal portion of Alx-4 as well as a proline-rich region downstream of the PL-homeodomain, but not the paired-tail at the C terminus. Taken together, these results demonstrate that Alx-4 is a potent transcriptional activator that is expressed at sites of epithelial-mesenchymal interactions during murine embryonic development (Hudson, 1998).

A new syndrome of X-linked myoclonic epilepsy with generalized spasticity and intellectual disability (XMESID) is described and the gene defect underlying this disorder is identified. A family is described in which six boys over two generations had intractable seizures as revealed using a validated seizure questionnaire, clinical examination, and EEG studies. Information on seizure disorders was obtained on 271 members of the extended family. Molecular genetic analysis included linkage studies and mutational analysis using a positional candidate gene approach. All six affected boys had myoclonic seizures and Tracheal cartilaginous sleeve (TCS is a congenital malformation characterized by fusion of the tracheal arches that may be isolated to a few tracheal arches, include the entire trachea, or extend beyond the carina into the bronchi); two had infantile spasms, but only one had hypsarrhythmia. EEG studies show diffuse background slowing with slow generalized spike wave activity. All affected boys had moderate to profound intellectual disability. Hyperreflexia was observed in obligate carrier women. A late-onset progressive spastic ataxia in the matriarch raises the possibility of late clinical manifestations in obligate carriers. The disorder was mapped to Xp11.2-22.2 with a maximum lod score of 1.8. A missense mutation (1058C>T/P353L) was identified within the homeodomain of the novel human Aristaless related homeobox gene (ARX). It is concluded XMESID is a rare X-linked recessive myoclonic epilepsy with spasticity and intellectual disability in boys. Hyperreflexia is found in carrier women. XMESID is associated with a missense mutation in ARX. This disorder is allelic with X-linked infantile spasms (ISSX; MIM 308350) where polyalanine tract expansions are the commonly observed molecular defect. Mutations of ARX are associated with a wide range of phenotypes; functional studies in the future may lend insights to the neurobiology of myoclonic seizures and infantile spasms (Scheffer, 2002).

Mental retardation and epilepsy often occur together. They are both heterogeneous conditions with acquired and genetic causes. Where causes are primarily genetic, major advances have been made in unraveling their molecular basis. The human X chromosome alone is estimated to harbor more than 100 genes that, when mutated, cause mental retardation. At least eight autosomal genes involved in idiopathic epilepsy have been identified, and many more have been implicated in conditions where epilepsy is a feature. Mutations have been identified in an X chromosome-linked, Aristaless-related, homeobox gene (ARX), in nine families with mental retardation (syndromic and nonspecific), various forms of epilepsy, including infantile spasms and myoclonic seizures, and dystonia. Two recurrent mutations, present in seven families, result in expansion of polyalanine tracts of the ARX protein. These probably cause protein aggregation, similar to other polyalanine and polyglutamine disorders. In addition, a missense mutation has been identified within the ARX homeodomain and a truncation mutation. Thus, it would seem that mutation of ARX is a major contributor to X-linked mental retardation and epilepsy (Stromme, 2002a).

Analyses were carried out on clinical data from 50 mentally retarded (MR) males in nine X-linked MR families, syndromic and non-specific, with mutations (duplication, expansion, missense, and deletion mutations) in the Aristaless related homeobox gene, ARX. Seizures were observed with all mutations and occurred in 29 patients, including one family with a novel myoclonic epilepsy syndrome associated with the missense mutation. Seventeen patients had infantile spasms. Other phenotypes included mild to moderate MR alone, or with combinations of dystonia, ataxia or autism. These data suggest that mutations in the ARX gene are important causes of MR, often associated with diverse neurological manifestations (Stromme, 2002b).

Investigation of a critical region for an X-linked mental retardation (XLMR) locus led to the identification of a novel Aristaless related homeobox gene (ARX). Inherited and de novo ARX mutations, including missense mutations and in frame duplications/insertions leading to expansions of polyalanine tracts in ARX, were found in nine familial and one sporadic case of MR. In contrast to other genes involved in XLMR, ARX expression is specific to the telencephalon and ventral thalamus. Notably there is an absence of expression in the cerebellum throughout development and also in adult. The absence of detectable brain malformations in patients suggests that ARX may have an essential role, in mature neurons, required for the development of cognitive abilities (Bienvenu, 2002).

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, ß-precursor, and Δ cells. Gene targeting in mouse embryonic stem cells has been used to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in ß-and Δ-cell numbers, whereas islet morphology remains intact. This study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on ß-and Δ-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), an accumulation of Pax4 and Arx transcripts has been demonstrated in Arx and Pax4 mutant mice, respectively. It is proposed that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts (Collombat, 2003).

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. The impact of Tbx15, Gli3, Alx4 and related genes was studied on the formation of the skeletal elements of the mouse shoulder and pelvic girdles. Severe reduction of the scapula is observed in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle (Kuijper, 2005).

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. This study reports crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein is expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice show severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb; (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors; (3) loss of tyrosine hydroxylase-positive periglomerular cells; (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons (Yashihara, 2005).

The specification of the different mouse pancreatic endocrine subtypes is determined by the concerted activities of transcription factors. However, the molecular mechanisms regulating endocrine fate allocation remain unclear. In the present study, the molecular consequences were uncovered of the simultaneous depletion of Arx and Pax4 activity during pancreas development. The findings reveal a so far unrecognized essential role of the paired-box-encoding Pax4 gene. Specifically, in the combined absence of Arx and Pax4, an early-onset loss of mature alpha- and ß-cells occurs in the endocrine pancreas, concomitantly with a virtually exclusive generation of somatostatin-producing cells. Furthermore, despite normal development of the PP-cells in the double-mutant embryos, an atypical expression of the pancreatic polypeptide (PP) hormone was observed in somatostatin-labelled cells after birth. Additional characterizations indicate that such an expression of PP is related to the onset of feeding, thereby unravelling an epigenetic control. Finally, the data provide evidence that both Arx and Pax4 act as transcriptional repressors that control one another's expression levels, thereby mediating proper endocrine fate allocation (Collombat, 2005).

Transcriptional regulation of Aristaless-related proteins

Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. The 5' regulatory region of the human Phox2a gene has been isolated and the transcriptional mechanisms underlying its expression have been studied. The minimal gene promoter was identified by means of molecular and functional criteria: its activity relies on a degenerate TATA box and a canonical Sp1 site. The region immediately upstream of the promoter stimulates transcription in a neurospecific manner because its deletion causes a substantial decline in reporter gene expression only in neuronal cells. This DNA region contains a putative binding site for homeodomain transcription factors, and its mutation severely affects the transcriptional activity of the entire 5' regulatory region, thus indicating that this site is necessary for the expression of Phox2a in this cellular context. The use of the electrophoretic mobility shift assay has shown that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrate that it is capable of transactivating the human Phox2a promoter. Many data obtained from knock-out mice support the hypothesis that Phox2a acts downstream of Phox2b during the development of most of the autonomic nervous system. The first molecular evidence has been provided that Phox2b can regulate the expression of Phox2a by directly binding to its 5' regulatory region (Flora, 2001).

Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming

Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve the ability to recapitulate cell differentiation events in vitro. This study demonstrates that Nkx2.2 is part of a large repression complex in pancreatic β cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts β-cell specification. Furthermore, Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in β cells. The Nkx2.2 TN mutation results in ectopic expression of Arx inβ cells, causing β-to-α-cell transdifferentiation. A corresponding β-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent β-to-α-cell reprogramming. Notably, subsequent removal of Arx in the β cells of Nkx2.2TNmut/TNmut mutant mice reverts the β-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in β cells are the primary regulatory events required for maintaining β-cell identity (Papizan, 2011).

Lhx6 directly regulates Arx and CXCR7 to determine cortical interneuron fate and laminar position

Cortical GABAergic interneurons have essential roles for information processing and their dysfunction is implicated in neuropsychiatric disorders. Transcriptional codes are elucidating mechanisms of interneuron specification in the MGE (a subcortical progenitor zone), which regulate their migration, integration, and function within cortical circuitry. Lhx6, a LIM-homeodomain transcription factor, is essential for specification of MGE-derived somatostatin and parvalbumin interneurons. This study demonstrates that some Lhx6-/- MGE cells acquire a CGE-like fate. Using an in vivo MGE complementation/transplantation assay, this study shows that Lhx6-regulated genes Arx and atypical chemokine receptor CXCR7 rescue divergent aspects of Lhx6-/- cell-fate and laminar mutant phenotypes and provide insight into a neonatal role for CXCR7 in MGE-derived interneuron lamination. Finally, Lhx6 directly binds in vivo to an Arx enhancer and to an intronic CXCR7 enhancer that remains active in mature interneurons. These data define the molecular identity of Lhx6 mutants and introduce technologies to test mechanisms in GABAergic interneuron differentiation (Vogt, 2014).

Aristaless-related proteins and development patterning

Continued: Evolutionary Homologs part 2/2


aristaless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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