Hox Complex Research Articles

Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11.

Mice with targeted disruptions in Hox genes have been generated to evaluate the role of the Hox complex in determining the mammalian body plan. This complex of 38 genes encodes transcription factors that specify regional information along the embryonic axes. Early in vertebrate evolution an ancestral complex shared with invertebrates was duplicated twice to give rise to the four linkage groups (Hox A, B, C and D). As a consequence, corresponding genes on the separate linkage groups, called paralogues, are most closely related to each other. Based on sequence similarities, the Hox genes have been subdivided into 13 paralogous groups. The five most 5' groups (Hox 9-13) pattern the posterior region of the vertebrate embryo and the appendicular skeleton. Mice with individual mutations in the paralogous genes hoxa-11 and hoxd-11 have been described. By breeding these two strains together we have generated double mutants which have dramatic phenotypes not apparent in mice homozygous for the individual mutations. The radius and the ulna of the forelimb are almost entirely eliminated, the axial skeleton shows homeotic transformations, and there are severe kidney defects not present in either single mutant. The limb and axial phenotypes are quantitative: as more mutant alleles are added to the genotype, the phenotype becomes progressively more severe. The appendicular skeleton defects suggest that paralogous Hox genes function together to specify limb outgrowth and patterning along the proximodistal axis.

Reciprocal Changes in Hox D13 and RAR-β2 Expression in Response to Retinoic Acid in Chick Limb Buds

The finding that retinoic acid (RA) has major effects on pattern formation in developing chick limbs has led to the conclusion that RA plays a central role in the normal development of the limb. In addition, it has been demonstrated that treatment with exogenous RA leads to changes in expression of homeobox-containing Hox complex genes within the developing limb. This has been used to argue that, since RA activates 5′ Hox D complex genes in the same temporal and spatial sequence as in normal development, Hox genes might be regulated by RA during normal limb development. In this study, we further examine the temporal and spatial changes in expression of Hox D13 and RAR-β2 transcripts in wing buds in response to local application of RA in vivo. We confirm reports that RAR-β2 expression is induced early and locally by RA. However, we find that the effect of RA on Hox D13 gene expression at the distal end of wing buds at stage 25/26 is downregulation of transcript expression. Furthermore, we find that activation of ectopic Hox D13 expression in response to implantation of RA beads along the anterior margin at stage 20/21 is indirect. Finally, the effects of RA exposure on Hox D13 expression appear to directly correlate with effects on the pattern of distal skeletal elements.

Setting limits on homeotic gene function: restraint of Sex combs reduced activity by teashirt and other homeotic genes.

Each of the homeotic genes of the HOM or HOX complexes is expressed in a limited domain along the anterior-posterior axis. Each homeotic protein directs the formation of characteristic structures, such as wings or ribs. In flies, when a heat shock-inducible homeotic gene is used to produce a homeotic protein in all cells of the embryo, only some cells respond by altering their fates. We have identified genes that limit where the homeotic gene Sex combs reduced (Scr) can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is prevented from inducing prothoracic structures by the three bithorax complex (BX-C) homeotic genes. However, two of the BX-C homeotic genes, Ultrabithorax (Ubx) and abdominal-A (abd-A), have no effect on the ability of Scr to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt (tsh) and in the last abdominal segment by the third BX-C gene, Abdominal-B (AbdB). Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function.

Developmental expression of the mouse Evx-2 gene: relationship with the evolution of the HOM/Hox complex

The mouse Evx-2 gene is located in the immediate vicinity of the Hoxd-13 gene, the most posteriorly expressed gene of the HOXD complex. While the Evx-1 gene is also physically linked to the HOXA complex, it is more distantly located from the corresponding Hoxa-13 gene. We have analysed the expression of Evx-2 during development and compared it to that of Evx-1 and Hoxd-13. We show that, even though Evx-2 is expressed in the developing CNS in a pattern resembling that of other Evx-related genes, the overall expression profile is similar to that of the neighbouring limbs and genitalia. We propose that the acquisition of expression features typical of Hox genes, together with the disappearance of some expression traits common to Evx genes, is due to the close physical linkage of Evx-2 to the HOXD complex, which results in Evx-2 expression being partly controlled by mechanisms acting in the HOX complex. This transposition of the Evx-2 gene next to the Hoxd-13 gene may have occurred soon after the large scale duplications of the HOX complexes. A scheme is proposed to account for the functional evolution of eve-related genes in the context of their linkage to the HOM/Hox complexes.

Positional information and pattern formation in development.

A widely used mechanism for pattern formation is based on positional information: cells acquire positional identities as in a coordinate system and then interpret this information according to their genetic constitution and developmental history. In Drosophila maternal factors establish the axes and set up a maternal system of positional information on which further patterning is built. There is a cascade of gene activity which leads both to the development of periodic structures, the segments, and to their acquiring a unique identity. This involves the binding of transcription factors to regulatory regions of genes to produce sharp thresholds. Many of the genes involved in these processes, particularly the Hox complex, are also involved in specifying the body axis and limbs of vertebrates. There are striking similarities in the mechanisms for specifying and recording positional identity in Drosophila and vertebrates.

The evolution of insect patterning mechanisms: a survey of progress and problems in comparative molecular embryology

ABSTRACT This report surveys data and interpretations presented by speakers in the Arthropod Session of the 1994 BSDB Spring Symposium. After a short review of phylogenetical aspects in premolecular insect embryology, the following topics are discussed: the ancestral germ type of pterygote insects, correlations between oogenesis and embryonic pattern formation, the universality or otherwise of bicoid as the anterior morphogen, novel functions in the insect Hox complex, the formal asymmetry between evolution and decay of complex gene networks, novel regulatory interactions as the main cause of evolutive changes, the repeated activity of conserved gene networks in successive steps of ontogenesis and strategies for future research. Interspersed are some unpublished data on oogenesis and pattern formation in lower dipterans, and their possible evolutionary implications.

The HOX complex neighbored by the EVX gene, as well as two other homeobox-containing genes, the GBX-class and the EN-class, are located on the same chromosomes 2 and 7 in humans

Two newly identified human homeobox-containing genes, GBX1 and GBX2, are closely related genes, as are members of the other homeobox genes, EN-1 and EN-2. GBX1 and EN-2 have been mapped to chromosome 7q36. The present study shows that GBX2 was mapped to chromosome 2q37. EN-1 was mapped to chromosome 2q14. Moreover, two HOX complexes neighbored by the EVX gene, HOXA and HOXD, are located at chromosome 7p15-p14 and 2q31-q37, respectively. Thus, it is possible that these homeobox genes were linked to each other on an ancestral genome and that the ancestral chromosome segment was duplicated during evolution.

Mice homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior transformations of the first and second cervical vertebrae, the atlas and the axis.

Gene targeting in embryo-derived stem (ES) cells was used to generate mice with a disruption in the homeobox-containing gene Hoxd-3 (Hox-4.1). Mice homozygous for this mutation show a radically remodeled craniocervical joint. The anterior arch of the atlas is transformed to an extension of the basioccipital bone of the skull. The lateral masses of the atlas also assume a morphology more closely resembling the exoccipitals and, to a variable extent, fuse with the exoccipitals. Formation of the second cervical vertebra, the axis, is also affected. The dens and the superior facets are deleted, and the axis shows 'atlas-like' characteristics. An unexpected observation is that different parts of the same vertebra are differentially affected by the loss of Hoxd-3 function. Some parts are deleted, others are homeotically transformed to more anterior structures. These observations suggest that one role of Hox genes may be to differentially control the proliferation rates of the mesenchymal condensations that give rise to the vertebral cartilages. Within the mouse Hox complex, paralogous genes not only encode very similar proteins but also often exhibit very similar expression patterns. Therefore, it has been postulated that paralogous Hox genes would perform similar roles. Surprisingly, however, no tissues or structures are affected in common by mutations in the two paralogous genes, Hoxa-3 and Hoxd-3.

Reconstruction from serial sections: A tool for developmental biology. Application to Hox genes expression in chicken wing buds

A method for analysing the localization of the Hox-4 gene complex by three-dimensional reconstruction of serial cross-sections is presented. It allows the simultaneous visualization of three Hox expression domains on the same reconstructed embryo and the study of the effect of a graft of chicken polarizing region on the de novo expression of the genes of the HOX-4 complex. The method consists of registering the sections, segmenting the biological patterns and finally displaying the reconstructed embryo. The precise relationships between the transcript domains of the three genes can thus be assessed in either the normal wing bud or upon pattern modification

Reconstruction from serial sections: A tool for developmental biology. Application to Hox genes expression in chicken wing buds

A method for analysing the localization of the Hox-4 gene complex by three-dimensional reconstruction of serial cross-sections is presented. It allows the simultaneous visualization of three Hox expression domains on the same reconstructed embryo and the study of the effect of a graft of chicken polarizing region on the de novo expression of the genes of the HOX-4 complex. The method consists of registering the sections, segmenting the biological patterns and finally displaying the reconstructed embryo. The precise relationships between the transcript domains of the three genes can thus be assessed in either the normal wing bud or upon pattern modification

Structural changes in the antennapedia complex of Drosophila pseudoobscura.

The discovery of the striking positional conservation between the Antennapedia and Bithorax homeotic gene complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters has had a substantial impact on our understanding of the evolution of development and its genetic regulation. Structural differences do exist among the mammalian Hox complexes and the ANT-C in D. melanogaster. To gain further insight into the evolutionary changes among these complexes, the ANT-C was cloned in the closely related species, Drosophila pseudoobscura. The overall structure of the ANT-C in D. pseudoobscura is highly similar to its D. melanogaster counterpart; however, two differences in the organization of the ANT-C have been identified. First, the z2 gene, a member of the ANT-C in D. melanogaster, is not present in the D. pseudoobscura ANT-C and is possibly absent from the D. pseudoobscura genome. Second, the orientation of the Deformed gene is inverted in D. pseudoobscura, providing it with a 5' to 3' direction of transcription identical to the remaining ANT-C homeobox genes with the exception of fushi tarazu. These differences demonstrate that subtle changes can occur in ANT-C structure during relatively short periods of evolutionary divergence, although the fundamental organization of the complex is conserved. These observations and others suggest that the complex is not absolutely rigid but that selective pressures have maintained this organization of genes for some functional reason that remains elusive.

Determination of trace aluminium by differential pulse adsorptive stripping voltammetry of aluminium(II) -8-hydroxyquinoline complex

A rapid and sensitive differential-pulse adsorptive stripping voltammetric method, based on the formation of an electroactive complex between aluminium(III) and 8-hydroxyquinoline (HOx), is presented for the determination of trace levels of aluminium. The complex showed an oxidative peak potential at +0.876 V vs. Ag/AgCl (saturated KCl) at a glassy carbon (GC) electrode in aqueous ammonium acetate (0.024 M, pH 6.8. A good linear relationship between the peak current and Al(III) concentration in the ranges 4.00 X 10 −8–5.00 X 10 −6 and 8.00 X 10 −6–4.00 X 10 −5 M were observed. In the lower Al(III) concentration region, the anodic peak was the result of the stripping of the absorbed Al(III)-HOx complex, whereas in the higher concentration range, the diffusional oxidation current of the solution species predominated. A surface saturation region was observed from 5.00 X 10 −6 to 8.00 X 10 −6 M Al(III). Relatively few interferences were found and these were easily masked. The detection limit for Al(III) for this method is 1.00 X 10 −8 M and ten determinations of Al(III) at 1.00 X 10 −7 M gave a relative standard deviation of 6.2%. The method was employed in the analysis of Al(III) in a US Environmental Protection Agency water pollution quality control sample (WP 386) and excellent agreement with the certified value was obtained.

Syndactylies and polydactylies: embryological overview and suggested classification.

In 1978, Temtamy and McKusick classified isolated, non-syndromic polydactyly and syndactyly, using a logical anatomical approach, into five distinct types for each group. Since then, there have been considerable advances in the molecular embryology of the developing limb bud. These include the proposal that retinoic acid and/or related retinoids are the morphogens responsible for the morphogenetic gradient giving rise to anterior-posterior pattern formation of the limb bud, the suggestion that the HOX4 complex and other homeotic genes may also be involved in patterning, and a greater understanding of other mechanisms such as programmed cell death in the shaping of the final hand and foot. This paper briefly reviews the molecular embryology of limb development and outlines the 'end-organ responsiveness' of the limbs to a variety of single-gene mutations. An alternative classification of syndactylies and polydactylies is suggested. It is still too early to match specific defects to individual genes with precision, and it is obvious that many important developmental genes remain to be identified; nevertheless, it is envisaged that clues from molecular embryological studies will become increasingly more useful.

Homeotic transformation of the occipital bones of the skull by ectopic expression of a homeobox gene.

Murine Hox genes have been postulated to play a role in patterning of the embryonic body plan. Gene disruption studies have suggested that for a given Hox complex, patterning of cell identity along the antero-posterior axis is directed by the more 'posterior' (having a more posterior rostral boundary of expression) Hox proteins expressed in a given cell. This supports the 'posterior prevalence' model, which also predicts that ectopic expression of a given Hox gene would result in altered structure only in regions anterior to its normal domain of expression. To test this model further, we have expressed the Hox-4.2 gene more rostrally than its normal mesoderm anterior boundary of expression, which is at the level of the first cervical somites. This ectopic expression results in a homeotic transformation of the occipital bones towards a more posterior phenotype into structures that resemble cervical vertebrae, whereas it has no effect in regions that normally express Hox-4.2. These results are similar to the homeotic posteriorization phenomenon generated in Drosophila by ectopic expression of genes of the homeotic complex HOM-C (refs 7-10; reviewed in ref. 3).

Effects of lithium chloride and retinoic acid on the expression of genes from the Xenopus laevis Hox 2 complex.

We show that Xenopus laevis has a Hox 2 complex, and that this complex is strongly conserved with the mammalian one, both in structure and in the rules of spatial and temporal sequential expression of its genes during the early stages of development. Lithium chloride and retinoic acid, two reagents known to alter axial patterning of the body when applied to Xenopus embryos, produce, respectively, embryos with reduced posterior but exaggerated anterior structures and embryos with truncation of anterior structures. We report here on the effect of these reagents on the expression of Hox 2 genes in the Xenopus embryos. LiCl has a dramatic effect on Hox genes, suppressing the expression of these genes during gastrulation and early neurulation. However, later on expression of these genes reaches significant levels, suggesting the existence of two phases in the control of Hox gene expression. Retinoic acid increases the steady state level of transcripts from Hox genes with the greatest effect on Hox 2.7, the most anterior of the genes studied. This suggests that the results obtained in EC cells (Simeone et al., 1990, 1991) reflect what occurs in vivo. Neither LiCl nor RA change the sequential order of the onset of expression of the genes, showing that these reagents do not perturb the molecular mechanisms used to establish the sequential activation of the genes of the Hox complexes.

The murine even-skipped-like gene Evx-2 is closely linked to the Hox-4 complex, but is transcribed in the opposite direction.

Note: Max-Planck-Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Gottingen, FRG. Reference UPDUB-ARTICLE-1992-001doi:10.1007/BF00355726 Record created on 2008-02-25, modified on 2017-05-12

The Hox-4.8 gene is localized at the 5′ extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development

We report the isolation and expression pattern of a novel mouse homeobox gene, Hox-4.8. Hox-4.8 is the most 5′-located homeobox gene in the HOX-4 complex. Sequence analysis confirmed that Hox-4.8 is a member of the subfamily of AbdominalB-related Hox-4 genes and revealed strong interspecies conservation. As for the human locus, Hox-4.8 is probably the last Hox gene in this part of the HOX-4 complex. During development, Hox-4.8 transcripts are restricted to the extremities of the embryonic anteroposterior axis and limbs as well as in the developing tail bud and to the most posterior segment of the gut (the rectum). Within the limb mesenchyme, Hox-4.8 is expressed in more posterodistal regions than those of its neighbour Hox-4.7. Hence, Hox-4.8 expression appears to be related to the last significant phenotypic changes towards the extremities of the embryonic body and limb axes.

HOX4 genes encode transcription factors with potential auto- and cross-regulatory capacities.

We have looked for the binding of several HOX4 complex homeoproteins in the genomic region containing the HOX4C promoter, between the human HOX4C and HOX4D genes. The HOX4C, HOX4D and Hox-4.3 homeoproteins bind to a phylogenetically highly conserved DNA fragment, which is located in the proximal part of this intergenic region and contains multiple binding sites for these HOX4 proteins. Using cotransfection experiments, we show that this endogenous DNA sequence can mediate transactivation by the HOX4D and HOX4C proteins and that this effect requires the presence of TAAT-related binding sites. The Hox-4.3 protein, in contrast, is unable to activate and can repress the activation observed with the two other proteins. These results show that the HOX4D and HOX4C genes are genuine sequence-specific transcription factors and suggest that, as in Drosophila, cross-regulatory interactions between these genes might be essential for their proper expression.

HOX-4 genes and the morphogenesis of mammalian genitalia.

We examined the temporal and spatial expression patterns of the homeo box HOX-4 complex genes during the morphogenesis of the genitalia of mice. The results show that only those Hox-4 genes that are expressed very posteriorly in the trunk, or very distally in the limbs, seem to be involved in the patterning of the genital tubercle. This is consistent with the idea of "temporal colinearity", which suggests that the very last structure to require patterning during vertebrate development will express Hox genes located at the 5' extremity of the HOX complexes. We also show that genital tubercle mesenchyme can respecify pattern in the chicken wing bud. This finding reinforces the concept of the uniformity in the patterning mechanisms along the various axes of the body.

Murine genes related to the Drosophila AbdB homeotic genes are sequentially expressed during development of the posterior part of the body.

The cloning, characterization and developmental expression patterns of two novel murine Hox genes, Hox-4.6 and Hox-4.7, are reported. Structural data allow us to classify the four Hox-4 genes located in the most upstream (5') position in the HOX-4 complex as members of a large family of homeogenes related to the Drosophila homeotic gene Abdominal B (AbdB). It therefore appears that these vertebrate genes are derived from a selective amplification of an ancestral gene which gave rise, during evolution, to the most posterior of the insect homeotic genes so far described. In agreement with the structural colinearity, these genes have very posteriorly restricted expression profiles. In addition, their developmental expression is temporally regulated according to a cranio-caudal sequence which parallels the physical ordering of these genes along the chromosome. We discuss the phylogenetic alternative in the evolution of genetic complexity by amplifying either genes or regulatory sequences, as exemplified by this system in the mouse and Drosophila. Furthermore, the possible role of 'temporal colinearity' in the ontogeny of all coelomic (metamerized) metazoans showing a temporal anteroposterior morphogenetic progression is addressed.