aristaless
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).
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).
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).
Aristaless-related proteins and development patterning Continued: Evolutionary Homologs part 2/2
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