Mouse Hox-2.2 specifies thoracic segmental identity in Drosophila embryos and larvae

The mouse genome has a number of homeobox genes that are structurally similar to the Drosophila Antenapedia (Antp) gene. We find that one of the mouse Antp-like genes, Hox-2.2, when expressed in developing Drosophila cells under control of a heat shock promoter, can induce homeotic transformations that are nearly identical to those caused by ectopic expression of Antp. In larvae, the Hox-2.2-induced transformations include thoracic denticle belts in place of head structures; in adults, the Hox-2.2 transformations include thoracic legs in place of antennae. The phenotypic effects of Hox-2.2 do not depend on the endogenous Antp gene, whose spatial limits of expression are unaffected by Hox-2.2 expression. Thus, in the Drosophila embryo, Hox-2.2 can substitute for some of the segmental identity functions of Antp, presumably by regulating the same set of downstream genes.     

The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes.

This paper reports the cloning of the fourth major murine homeogene complex, HOX-5. The partial characterization of this gene cluster revealed the presence of two novel genes (Hox-5.2, Hox-5.3) located at the 5' extremity of this complex. In situ hybridization experiments showed that these two genes are transcribed in very posterior domains during embryonic and foetal development. We also show that Hox-1.6, the gene located at the 3' most position in the HOX-1 complex, has a very anterior expression boundary during early development. These results clearly support the recently proposed hypothesis that the expression of murine Antp-like homeobox-containing genes along the antero-posterior developing body axis follows a positional hierarchy which reflects their respective physical positions within the HOX clusters, similar to that which is found for the Drosophila homeotic genes. Such a structural and functional organization is likely conserved in most vertebrates. Moreover, on the basis of sequence comparisons, we propose that the ordering of homeobox-containing genes within clusters has been conserved between Drosophila and the house mouse. Thus, very different body plans might be achieved, both in insects and vertebrates, by evolutionarily conserved gene networks possibly displaying similar regulatory interactions.     

Gene organization of murine homeobox-containing gene clusters

A chromosomal walk which links a previously described and a new homeobox to the Hox-2 murine homeobox gene cluster is described, and the nucleotide sequence of the new homeobox is presented. With these new data the Hox-2 gene cluster contains seven loci on an approximately 100-kb-long physical map. Homology comparisons reveal that a significant number of vertebrate homeoboxes are in fact analogous. We also find that the linear order of homologous homeoboxes is similar in the two murine gene complexes, Hox-1 and Hox-2, and among the human homeobox loci on chromosome 17. Conservation of the homeo-domain and the linear gene order of homeobox-containing genes in vertebrates is discussed in light of the interactions and the anteroposterior linear order of homeotic loci in insects.     

An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution.

The embryology of amphioxus has much in common with vertebrate embryology, reflecting a close phylogenetic relationship between the two groups. Amphioxus embryology is simpler in several key respects, however, including a lack of pronounced craniofacial morphogenesis. To gain an insight into the molecular changes that accompanied the evolution of vertebrate embryology, and into the relationship between the amphioxus and vertebrate body plans, we have undertaken the first molecular level investigation of amphioxus embryonic development. We report the cloning, complete DNA sequence determination, sequence analysis and expression analysis of an amphioxus homeobox gene, AmphiHox3, evolutionarily homologous to the third-most 3' paralogous group of mammalian Hox genes. Sequence comparison to a mammalian homologue, mouse Hox-2.7 (HoxB3), reveals several stretches of amino acid conservation within the deduced protein sequences. Whole mount in situ hybridization reveals localized expression of AmphiHox3 in the posterior mesoderm (but not in the somites), and region-specific expression in the dorsal nerve cord, of amphioxus neurulae, later embryos and larvae. The anterior limit to expression in the nerve cord is at the level of the four/five somite boundary at the neurula stage, and stabilises to just anterior to the first nerve cord pigment spot to form. Comparison to the anterior expression boundary of mouse Hox-2.7 (HoxB3) and related genes suggests that the vertebrate brain is homologous to an extensive region of the amphioxus nerve cord that contains the cerebral vesicle (a region at the extreme rostral tip) and extends posterior to somite four.(ABSTRACT TRUNCATED AT 250 WORDS)     

The murine and Drosophila homeobox gene complexes have common features of organization and expression

In situ hybridization analysis of mouse embryos shows the seven members of the Hox-2 complex to be differentially expressed in the central and peripheral nervous system and in mesodermal derivatives (somites and lung). Beginning at the 5' end of the cluster, each successive gene displays a more anterior boundary of expression in the central nervous system. A gene's position in the Hox-2 cluster therefore reflects its relative domain of expression along the anteroposterior axis of the embryo, a feature observed with Drosophila homeotic genes. Sequence comparisons of the Hox-2 cluster with other mouse and Drosophila homeobox genes have defined subgroups of related genes; in the mouse there are four clusters related by duplication and divergence. Alignment shows a clear relationship among genes in the mouse and Drosophila complexes, based on relative position, sequence identity, and domains of expression along the rostral-caudal axis. Our results argue that these complexes arose from a common ancestor, present before the divergence of lineages that gave rise to arthropods and vertebrates.     

Expression of Hox-2.4 homeobox gene directed by proviral insertion in a myeloid leukemia.

The presence of an altered Hox-2.4 gene in the WEHI3B murine myeloid leukemia suggests that homeobox genes may contribute to neoplasia. A survey of 31 leukemia cell lines of the myeloid, lymphoid and erythroid lineages revealed that Hox-2.4 was expressed only in WEHI3B and the pre-B lymphoid line 70Z/3, in which no DNA rearrangement was observed. To clarify the WEHI3B alteration and normal Hox-2.4 structure, we have sequenced near full length cDNA clones from WEHI3B and 70Z/3, and the 5' portion of the normal Hox-2.4 gene. A WEHI3B cDNA clone demonstrates that an intracisternal A-particle (IAP) provirus has inserted within the first exon of the gene and generated a Hox-2.4 mRNA with a 5' sequence derived from the IAP long terminal repeat. A remarkable degree of similarity found between the amino acid sequences of Hox-2.4 and Hox-3.1, which reside on different chromosomes, supports the notion that an ancient homeobox gene cluster has been duplicated and dispersed early in vertebrate evolution.     

Dominant mutation of the murine Hox-2.2 gene results in developmental abnormalities.

Genes carrying the homeobox were originally identified in Drosophila, in which they are now known to play key roles in establishing segmentation patterns and in determining segment identities. A number of genes with striking homology to the Drosophila homeobox genes have now been found in the mouse genome, and mutational analysis is beginning to shed light on their function in mammalian development. To understand better the developmental significance of the murine Hox-2.2 gene, we have generated gain of function mutants by using the chicken beta-actin promoter to drive ubiquitous expression in transgenic mice. The resulting Hox-2.2 misexpression produces early postnatal lethality as well as craniofacial and axial skeletal perturbations that include open eyes at birth, cleft palate, micrognathia, microtia, skull bone deficiencies, and structural and positional alterations in the vertebral column. We repeatedly observe complete or partial absence of the supraoccipital bone and malformations of the exoccipital and the basioccipital bones. These results suggests a role for the Hox-2.2 gene in specifying positional identity along the anterior-posterior axis.     

DNA rearrangement of a homeobox gene in myeloid leukaemic cells.

A homeobox gene rearrangement has been detected in WEHI-3B mouse myeloid leukaemic cells. The rearranged gene was identified as Hox-2.4 which is a member of the Hox-2 gene cluster on mouse chromosome 11. Both the normal and the rearranged genes were cloned and analysed, and the rearranged genomic Hox-2.4 gene was sequenced. The results indicate that the rearrangement is due to insertion of an intracisternal A particle 5' upstream to Hox-2.4 and that this resulted in constitutive expression of the homeobox gene. It is suggested that constitutive expression of the homeobox gene may interrupt the normal development program in these leukaemic cells.     

Molecular cloning and characterization of homeo-box-containing genes from Atlantic salmon

As the most primitive group among vertebrates, fish might serve as a model system when studying the genetic regulation of embryogenesis in higher animals. To identify genes important for early development, we have constructed a genomic library from Atlantic salmon (Salmo salar) and screened it with homeobox-containing probes from Drosophila melanogaster. Five different salmon homeoboxes were isolated. Two of these were located in the same clone, separated by only 7.5 kb. This demonstrates the presence of clustered homeobox genes in fish. The two clustered homeoboxes were sequenced and shown to be closely related to the ANT-C/BX-C class of Drosophila, being about 80% homologous to the Ultrabithorax gene (Ubx) homeobox. One of the clustered genes appears to be the salmon equivalent of the mouse Hox-2.1 gene, indicating that some of the vertebrate homeobox-containing genes are conserved in evolution. A more diverged homeobox that shares only 60% homology with Ubx, was also sequenced. In analogy to Drosophila, therefore, the salmon genome contains more than one class of homeoboxes. In addition, Northern-blot experiments demonstrated that two of the homeobox genes are expressed in salmon embryos, suggesting their importance for proper development.     

Hox-3.6: isolation and characterization of a new murine homeobox gene located in the 5' region of the Hox-3 cluster.

Most members of the murine Hox gene system can be grouped into two subclasses based on their structural similarity to either one of the Drosophila homeotic genes Antennapedia (Antp) or Abdominal B (AbdB). All the AbdB-like genes reported thus far are located in the 5' region of their respective cluster. We describe here the isolation, structural characterization and spatio-temporal expression pattern of a new AbdB-like homeobox gene designated Hox-3.6 that is located in the 5' region of the Hox-3 cluster. Hox-3.6 has an extreme posterior expression domain in embryos of 12.5 days of gestation, a feature that has thus far only been observed for the 5' most genes of the Hox-4 cluster. Like the other members of the AbdB subfamily, Hox-3.6 exhibits spatially restricted expression in the hindlimb bud, but the expression domain is antero-proximal in contrast to the postero-distal domain reported for its cognate gene Hox-4.5. Structural analysis of the 5' region revealed the presence of a 35 bp sequence which shares homology and relative 5' position with an upstream sequence present in its two nearest downstream neighbors, Hox-3.2 and -3.1.     

Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression.

Homeobox genes play a key role in specifying the segmented body plan of Drosophila, and recent work suggests that at least several homeobox genes may play a regulatory role during vertebrate limb morphogenesis. We have used degenerate oligonucleotide primers from highly conserved domains in the homeobox motif to amplify homeobox gene segments from chick embryo limb bud cDNAs using the polymerase chain reaction. Expression of a large number of homeobox genes (at least 17) is detected using this approach. One of these genes contains a novel homeobox loosely related to the Drosophila Abdominal B class, and was further analyzed by determining its complete coding sequence and evaluating its expression during embryogenesis by in situ hybridization. Based on sequence and expression patterns, we have designated this gene as Ghox 4.7 and believe that it is the chick homologue of the murine Hox 4.7 gene (formerly Hox 5.6). Ghox 4.7 is expressed primarily in limb buds during development and shows a striking spatial restriction to the posterior zone of the limb bud, suggesting a role in specifying anterior-posterior pattern formation. In chick, this gene also displays differences in expression between wing and leg buds, raising the possibility that it may participate in specifying limb-type identity.     

Inhibition of specific pathways of myeloid cell differentiation by an activated Hox-2.4 homeobox gene.

Abnormal expression of homeobox genes is one of the abnormalities associated with the development of murine and human leukemia. Myeloid leukemic cells that can be induced to differentiate to mature cells by interleukin 6 were stably transfected with an activated Hox-2.4 homeobox gene. Expression of the Hox-2.4 gene in the transfected clones inhibited specific pathways of the myeloid differentiation program induced by interleukin 6. The expression of some genes associated with differentiation was almost completely blocked, and the expression of other genes was either partially inhibited or not affected. The results support the hypothesis that abnormal expression of Hox-2.4 may contribute to the development of leukemia by interfering with the differentiation program.