Hox Gene Function Research Articles

Hox and wings

In many bilaterian phyla, appendages are morphological traits that characterise the identity of the various body parts. In pterygote insects, wings are dorsal appendages on the thorax. The famous "bithorax" fly created by Ed Lewis is the emblematic example of the role of Hox genes.1 Now, Tomoyasu et al.,2 using classical genetics, transgenesis and RNAi, have examined the function of thoracic Hox genes in the beetle Tribolium castaneum. Beetles have rigid elytra in place of the first pair of wings. Instead of the expected transformation of the elytron into a wing, loss of Hox genes' function leads to the homeotic transformation of the second pair of dorsal appendages, the wings, into elytra. This has important consequences for the way that we see the role of Hox genes in development and evolution.

Hox genes and their candidate downstream targets in the developing central nervous system.

1. Homeobox (Hox) genes were originally discovered in the fruit fly Drosophila, where they function through a conserved homeodomain as transcriptional regulators to control embryonic morphogenesis. Since then over 1000 homeodomain proteins have been identified in several species. In vertebrates, 39 Hox genes have been identified as homologs of the original Drosophila complex, and like their Drosophila counterparts they are organized within chromosomal clusters. Vertebrate Hox genes have also been shown to play a critical role in embryonic development as transcriptional regulators. 2. Both the Drosophila and vertebrate Hox genes have been shown to interact with various cofactors, such as the TALE homeodomain proteins, in recognition of consensus sequences within regulatory elements of their target genes. These protein-protein interactions are believed to contribute to enhancing the specificity of target gene recognition in a cell-type or tissue- dependent manner. The regulatory activity of a particular Hox protein on a specific regulatory element is highly variable and dependent on its interacting partners within the transcriptional complex. 3. In vertebrates, Hox genes display spatially restricted patterns of expression within the developing CNS, both along the anterioposterior and dorsoventral axis of the embryo. Their restricted gene expression is suggestive of a regulatory role in patterning of the CNS, as well as in cell specification. Determining the precise function of individual Hox genes in CNS morphogenesis through classical mutational analyses is complicated due to functional redundancy between Hox genes. 4. Understanding the precise mechanisms through which Hox genes mediate embryonic morphogenesis requires the identification of their downstream target genes. Although Hox genes have been implicated in the regulation of several pathways, few target genes have been shown to be under their direct regulatory control. Development of methodologies used for the isolation of target genes and for the analysis of putative targets will be beneficial in establishing the genetic pathways controlled by Hox factors. 5. Within the developing CNS various cell adhesion molecules and signaling molecules have been identified as candidate downstream target genes of Hox proteins. These targets play a role in processes such as cell migration and differentiation, and are implicated in contributing to neuronal processes such as plasticity and/or specification. Hence, Hox genes not only play a role in patterning of the CNS during early development, but may also contribute to cell specification and identity.

Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function

Vertebrate HoxA and HoxD cluster genes are required for proper limb development. However, early lethality, compensation and redundancy have made a full assessment of their function difficult. Here we describe mice that are lacking all Hoxa and Hoxd functions in their forelimbs. We show that such limbs are arrested early in their developmental patterning and display severe truncations of distal elements, partly owing to the absence of Sonic hedgehog expression. These results indicate that the evolutionary recruitment of Hox gene function into growing appendages might have been crucial in implementing hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages. Accordingly, these mutant limbs may be reminiscent of an ancestral trunk extension, related to that proposed for arthropods.

Retinaldehyde dehydrogenase 2 and Hoxc8 are required in the murine brachial spinal cord for the specification of Lim1+ motoneurons and the correct distribution of Islet1+ motoneurons

Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.

The pathophysiology of HOX genes and their role in cancer

The HOM-C clustered prototype homeobox genes of Drosophila, and their counterparts, the HOX genes in humans, are highly conserved at the genomic level. These master regulators of development continue to be expressed throughout adulthood in various tissues and organs. The physiological and patho-physiological functions of this network of genes are being avidly pursued within the scientific community, but defined roles for them remain elusive. The order of expression of HOX genes within a cluster is co-ordinated during development, so that the 3' genes are expressed more anteriorly and earlier than the 5' genes. Mutations in HOXA13 and HOXD13 are associated with disorders of limb formation such as hand-foot-genital syndrome (HFGS), synpolydactyly (SPD), and brachydactyly. Haematopoietic progenitors express HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cells. In leukaemia, dysregulated HOX gene expression can occur due to chromosomal translocations involving upstream regulators such as the MLL gene, or the fusion of a HOX gene to another gene such as the nucleoporin, NUP98. Recent investigations of HOX gene expression in leukaemia are providing important insights into disease classification and prediction of clinical outcome. Whereas the oncogenic potential of certain HOX genes in leukaemia has already been defined, their role in other neoplasms is currently being studied. Progress has been hampered by the experimental approach used in many studies in which the expression of small subsets of HOX genes was analysed, and complicated by the functional redundancy implicit in the HOX gene system. Attempts to elucidate the function of HOX genes in malignant transformation will be enhanced by a better understanding of their upstream regulators and downstream target genes.

Ectopic expression of Hoxd10 in thoracic spinal segments induces motoneurons with a lumbosacral molecular profile and axon projections to the limb

Hox genes encode anterior-posterior identity during central nervous system development. Few studies have examined Hox gene function at lumbosacral (LS) levels of the spinal cord, where there is extensive information on normal development. Hoxd10 is expressed at high levels in the embryonic LS cord but not the thoracic cord. To test the hypothesis that restricted expression of Hoxd10 contributes to the attainment of an LS identity, and specifically an LS motoneuron identity, Hoxd10 was ectopically expressed in thoracic segments in chick embryos by means of in ovo electroporation. Regional motoneuron identity was assessed after the normal period of motoneuron differentiation. Subsets of motoneurons in transfected thoracic segments developed a molecular profile normally shown by LS motoneurons, including Lim 1 and RALDH2 expression. In addition, motoneurons in posterior thoracic segments showed novel axon projections to two muscles in the anterodorsal limb, the sartorius and anterior iliotibialis muscles. At thoracic levels, we also found a decrease in motoneuron numbers and a reduction in gonad size. These last findings suggest that early and high levels of Hox expression impeded motoneuron development and neural-mesodermal interactions. Despite these adverse effects, our data indicate that Hoxd10 expression is sufficient to induce LS motoneuron identity and axon trajectories characteristic of motoneurons in the LS region.

In vivo mutagenesis of the Hoxb8 hexapeptide domain leads to dominant homeotic transformations that mimic the loss-of-function mutations in genes of the Hoxb cluster

Hox proteins are transcription factors that control developmental pathways along the anteroposterior axis of vertebrates. On their own, Hox proteins bind DNA weakly, but they gain specificity and affinity by interaction with members of the PBC subfamily of homeobox proteins. In vitro studies indicate that most of these interactions are mediated by the conserved hexapeptide motif of the Hox proteins. To study the significance of these interactions in vivo, we have generated mice that carry mutations in the Hoxb8 hexapeptide motif. Analysis of skeletal features of these mice reveals the presence of a dominant phenotype consisting of homeotic transformations, similar to those observed in mice with a loss-of-function of Hox genes, such as Hoxa7, Hoxb7, and Hoxb9. Genetic tests demonstrate that the mutations in the Hoxb8 hexapeptide motif are affecting the function of other genes located in the Hoxb cluster. The expression pattern of these genes is not affected; rather it appears that the mutant Hoxb8 protein interferes with the function of other Hox genes by binding to their targets. Our findings suggest that the homeotic transformations result from altered DNA binding specificity of the mutant Hoxb8 protein, implicating the cooperative binding between Hoxb8 hexapeptide motif and cofactors as a critical element in the fine-tuning of Hoxb8 protein target specificity. This is the first time the function of the hexapeptide domain has been evaluated in vivo in mouse development.

In vivo mutagenesis of the Hoxb8 hexapeptide domain leads to dominant homeotic transformations that mimic the loss-of-function mutations in genes of the Hoxb cluster

Hox proteins are transcription factors that control developmental pathways along the anteroposterior axis of vertebrates. On their own, Hox proteins bind DNA weakly, but they gain specificity and affinity by interaction with members of the PBC subfamily of homeobox proteins. In vitro studies indicate that most of these interactions are mediated by the conserved hexapeptide motif of the Hox proteins. To study the significance of these interactions in vivo, we have generated mice that carry mutations in the Hoxb8 hexapeptide motif. Analysis of skeletal features of these mice reveals the presence of a dominant phenotype consisting of homeotic transformations, similar to those observed in mice with a loss-of-function of Hox genes, such as Hoxa7, Hoxb7, and Hoxb9 . Genetic tests demonstrate that the mutations in the Hoxb8 hexapeptide motif are affecting the function of other genes located in the Hoxb cluster. The expression pattern of these genes is not affected; rather it appears that the mutant Hoxb8 protein interferes with the function of other Hox genes by binding to their targets. Our findings suggest that the homeotic transformations result from altered DNA binding specificity of the mutant Hoxb8 protein, implicating the cooperative binding between Hoxb8 hexapeptide motif and cofactors as a critical element in the fine-tuning of Hoxb8 protein target specificity. This is the first time the function of the hexapeptide domain has been evaluated in vivo in mouse development.

Establishing A–P Polarity in the Embryonic Heart Tube: A Conserved Function of Hox Genes in Drosophila and Vertebrates?

The correct establishment of anterior–posterior (A–P) polarity in the vertebrate embryonic heart tube during embryogenesis is crucial for the proper morphogenesis of the mature heart, but the molecular details of this process are poorly understood. Elucidation of this process should be facilitated by findings of recent studies regarding the establishment of A–P polarity in the Drosophila equivalent of the heart, the dorsal vessel. These studies have demonstrated that members of the Drosophila homeotic selector (Hox) gene family play important roles in this process. It appears that the homeotic gene abdominal-A is a key determinant of A–P polarity in the dorsal vessel, due to its function in specifying the posterior chamber of the dorsal vessel, and that other homeotic genes may function in specifying more anterior portions of the dorsal vessel. Another role of certain homeotic genes is to determine the extent of the cardiogenic region within the early embryonic mesoderm. These results suggest that the Hox genes may also play some role in the positioning of the embryonic heart field and the determination of A–P polarity in the vertebrate embryonic heart tube.

Perturbed thyroid morphology and transient hypothyroidism symptoms in Hoxa5 mutant mice.

The Hox family of transcriptional regulators has been extensively studied for their role in axial and appendicular patterning. Genetic analyses have also unveiled Hox gene function in organogenesis and postnatal development. A phenotypical survey of the Hoxa5(-/-) mutant mice shows that the surviving mutants display symptoms of hypothyroidism, including transient growth retardation, and delayed eye opening and ear elevation. Thyroid gland morphogenesis initiates normally, but follicle formation and thyroglobulin processing are abnormal at late gestation. The expression of several molecular markers essential for thyroid gland formation and function, namely Nkx2.1, Pax8, and Titf2, is affected in the developing thyroid gland of Hoxa5(-/-) mutants. As a consequence, the expression of thyroid effector genes, including the thyroglobulin and thyroperoxidase genes, is perturbed. Our characterization reveals that the loss of Hoxa5 function transiently affects thyroid development in a non-cell autonomous manner.

Extreme conservation of non-repetitive non-coding regions near HoxDcomplex of vertebrates

Homeotic gene complexes determine the anterior-posterior body axis in animals. The expression pattern and function of hox genes along this axis is colinear with the order in which they are organized in the complex. This 'chromosomal organization and functional correspondence' is conserved in all bilaterians investigated. Although the molecular basis of this 'colinearity' in not yet understood, it is possible that there are control elements within or in the proximity of these complexes that establish and maintain the expression patterns of hox genes in a coordinated fashion. We report here an unprecedented conservation of non-coding DNA sequences adjacent to the HoxD complex of vertebrates. Stretches of hundreds of base pairs in a 7 kb region, upstream of HoxD complex, show 100% conservation from fish to human. Using primers designed from these sequences of human HoxD complex, we amplified the corresponding regions from different vertebrates, including mammals, aves, reptiles, amphibians and pisces. Such a high degree of conservation, where no variation was allowed during ~500 million years of evolution, suggests critical function for these sequences in the regulation of the HoxD complex. Furthermore, these sequences provide a molecular handle to gain insight into the mechanism of regulation of this complex.

Integrative biology and the developing limb bud.

The identification or selective construction of mutations within genes has allowed researchers to explore the downstream effects of gene disruption. Although these approaches have been successful, a limitation in our assessment of the consequences of conditional changes, and thereby our understanding of roles or function of genes, limits the degree to which we examine the effects of our manipulations. It is also clear that linear associations are incorrect models for describing development, and newer methods now give us an opportunity to practice an integrative biology. In our attempts to explore the consequences of Hoxa13 disruption in mice and humans, it has become clear that a better understanding of the consequences of gene alteration may be achievable by taking a broader approach with a long-term view. Fundamental questions regarding Hox gene function in vertebrates, including those related to the number of target genes; the degree of overlap of target gene regulation among paralogs; the magnitude of modulation exerted; and the identity of genes that are activated versus repressed need to be explored if a more thorough mechanistic understanding is to be achieved. To begin to address these questions, we undertook a comprehensive analysis of the expression of genes within developing limb buds of mice, and here we present some of our preliminary results. Our efforts will further (1) the exploration of the broader genetic relationships of expressed genes, (2) the determination of parallels or variations in target usage for a given gene in different tissues and between different organisms, (3) the evaluation of limb patterning mechanisms in other animal model systems, and (4) the exploration of gene expression hierarchies regulated by HOX proteins in developmental systems.

The Hox Paradox: More Complex(es) Than Imagined

An understanding of the origin of different body plans requires knowledge of how the genes and genetic pathways that control embryonic development have evolved. The Hox genes provide an appealing starting point for such studies because they play a well-understood causal role in the regionalization of the body plan of all bilaterally symmetric animals. Vertebrate evolution has been characterized by gene, and possibly genome, duplication events, which are believed to have provided raw genetic material for selection to act upon. It has recently been established that the Hox gene organization of ray-finned fishes, such as the zebrafish, differs dramatically from that of their lobe-finned relatives, a group that includes humans and all the other widely used vertebrate model systems. This unusual Hox gene organization of zebrafish is the result of a duplication event within the ray-finned fish lineage. Thus, teleosts, such as zebrafish, have more Hox genes arrayed over more clusters (or “complexes”) than do tetrapod vertebrates. Here, I review our understanding of Hox cluster architecture in different vertebrates and consider the implications of gene duplication for Hox gene regulation and function and the evolution of different body plans.

Tribolium Hox genes repress antennal development in the gnathos and trunk

Evidence from Drosophila suggests that Hox genes not only specify regional identity, but have the additional function of repressing antennal development within their normal domains. This is dramatically demonstrated by a series of Hox mutants in the red flour beetle, Tribolium castaneum, and is likely an ancient function of Hox genes in insetcs.

A green fluorescent protein reporter genetic screen that identifies modifiers of Hox gene function in the Drosophila embryo.

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. In this study, we describe a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. We found 15 deficiencies that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways, which suggests an extensive use of signaling in the control of Hox gene function.

Functional Comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 Homeoboxes

A number of models attempt to explain the functional relationships of Hox genes. The functional equivalence model states that mammalian Hox-encoded proteins are largely functionally equivalent, and that Hox quantity is more important than Hox quality. In this report, we describe the results of two homeobox swaps. In one case, the homeobox of Hoxa 11 was replaced with that of the very closely related Hoxa 10. Developmental function was assayed by analyzing the phenotypes of all possible allele combinations, including the swapped allele, and null alleles for Hoxa 11 and Hoxd 11. This chimeric gene provided wild-type function in the development of the axial skeleton and male reproductive tract, but served as a hypomorph allele in the development of the appendicular skeleton, kidneys, and female reproductive tract. In the other case, the Hoxa 11 homeobox was replaced with that of the divergent Hoxa 4 gene. This chimeric gene provided near recessive null function in all tissues except the axial skeleton, which developed normally. These results demonstrate that even the most conserved regions of Hox genes, the homeoboxes, are not functionally interchangeable in the development of most tissues. In some cases, developmental function tracked with the homeobox, as previously seen in simpler organisms. Homeoboxes with more 5′ cluster positions were generally dominant over more 3′ homeoboxes, consistent with phenotypic suppression seen in Drosophila. Surprisingly, however, all Hox homeoboxes tested did appear functionally equivalent in the formation of the axial skeleton. The determination of segment identity is one of the most evolutionarily ancient functions of Hox genes. It is interesting that Hox homeoboxes are interchangeable in this process, but are functionally distinct in other aspects of development.

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Consequences of Hox gene duplication in the vertebrates: an investigation of the zebrafish Hox paralogue group 1 genes.

As a result of a whole genome duplication event in the lineage leading to teleosts, the zebrafish has seven clusters of Hox patterning genes, rather than four, as described for tetrapod vertebrates. To investigate the consequences of this genome duplication, we have carried out a detailed comparison of genes from a single Hox paralogue group, paralogue group (PG) 1. We have analyzed the sequences, expression patterns and potential functions of all four of the zebrafish PG1 Hox genes, and compared our data with that available for the three mouse genes. As the basic functions of Hox genes appear to be tightly constrained, comparison with mouse data has allowed us to identify specific changes in the developmental roles of Hox genes that have occurred during vertebrate evolution. We have found variation in expression patterns, amino acid sequences within functional domains, and potential gene functions both within the PG1 genes of zebrafish, and in comparison to mouse PG1 genes. We observed novel expression patterns in the midbrain, such that zebrafish hoxa1a and hoxc1a are expressed anterior to the domain traditionally thought to be under Hox patterning control. The hoxc1a gene shows significant coding sequence changes in known functional domains, which correlate with a reduced capacity to cause posteriorizing transformations. Moreover, the hoxb1 duplicate genes have differing functional capacities, suggesting divergence after duplication. We also find that an intriguing function 'shuffling' between paralogues has occurred, such that one of the zebrafish hoxb1 duplicates, hoxb1b, performs the role in hindbrain patterning played in mouse by the non-orthologous Hoxa1 gene.

Hox and ParaHox Genes in Flatworms: Characterization and Expression1

Flatworms (phylum Platyhelminthes) are favourite organisms in Developmental Biology and Zoology because of their extraordinary powers of regeneration and because they may hold a pivotal place in the origin and evolution of the Bilateria. Hox genes play key roles in both processes: setting up the new anteroposterior pattern in the former, and as qualitative markers of phylogenetic affinities among bilaterian phyla in the latter. We have searched for Hox and ParaHox genes in several flatworm groups spanning from freshwater triclads to marine polyclads and, more recently, in the acoels, the likely earliest extant bilaterian. We have isolated and sequenced eight Hox genes from the freshwater triclad Girardia tigrina and three Hox and two ParaHox genes from the polyclad Discocelis tigrina. Data from the acoels Paratomella rubra and Convoluta roscoffensis is also reported. Flatworm Hox sequences and 18S rDNA sequence data support clear affinities of Platyhelminthes to spiralian lophotrochozoans. The basal position of acoel flatworms supported from recent 18S rDNA data, remains still uncertain. Expression of Hox genes in intact and regenerating adult organisms show nested patterns with graded anterior expression boundaries, or ubiquitous expression. New approaches to study the function of Hox genes in flatworms, such as RNA interference are briefly discussed.

Hox genes and the phylogeny of the arthropods.

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla - chelicerates, crustaceans, hexapods, and myriapods - are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2-9]. A cluster of regulatory genes, the Hox genes, control segment identity in arthropods, and comparisons of the sequences and functions of Hox genes can reveal evolutionary relationships [10]. We used Hox gene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.