A low diversity of ANTP class homeobox genes in Placozoa

Homeobox genes of the ANTP and PRD classes play important roles in body patterning of metazoans, and a large diversity of these genes have been described in bilaterian animals and cnidarians. Trichoplax adhaerens (Phylum Placozoa) is a small multicellular marine animal with one of the simplest body organizations of all metazoans, showing no symmetry and a small number of distinct cell types. Only two ANTP class genes have been described from Trichoplax: the Hox/ParaHox gene Trox-2 and a gene related to the Not family. Here we report an extensive screen for ANTP class genes in Trichoplax, leading to isolation of three additional ANTP class genes. These can be assigned to the Dlx, Mnx and Hmx gene families. Sequencing approximately 12-20 kb around each gene indicates that none are part of tight gene clusters, and in situ hybridization reveals that at least two have spatially restricted expression around the periphery of the animal. The low diversity of ANTP class genes isolated in Trichoplax can be reconciled with the low anatomical complexity of this animal, although the finding that these genes are assignable to recognized gene families is intriguing.     

Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes

Homeobox genes encode DNA-binding proteins, many of which are implicated in the control of embryonic development. Evolutionarily, most homeobox genes fall into two related clades: the ANTP and the PRD classes. Some genes in ANTP class, notably Hox, ParaHox, and NK genes, have an intriguing arrangement into physical clusters. To investigate the evolutionary history of these gene clusters, we examined homeobox gene chromosomal locations in the cephalochordate amphioxus, Branchiostoma floridae. We deduce that 22 amphioxus ANTP class homeobox genes localize in just three chromosomes. One contains the Hox cluster plus AmphiEn, AmphiMnx, and AmphiDll. The ParaHox cluster resides in another chromosome, whereas a third chromosome contains the NK type homeobox genes, including AmphiMsx and AmphiTlx. By comparative analysis we infer that clustering of ANTP class homeobox genes evolved just once, during a series of extensive cis-duplication events of genes early in animal evolution. A trans-duplication event occurred later to yield the Hox and ParaHox gene clusters on different chromosomes. The results obtained have implications for understanding the origin of homeobox gene clustering, the diversification of the ANTP class of homeobox genes, and the evolution of animal genomes.     

A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.     

Convergent evolution of clustering of Iroquois homeobox genes across metazoans

Vertebrate and Drosophila Iroquois genes are organized in clusters of 3 genes sharing blocks of conserved regulatory sequences. Here, we report a 3-gene cluster in the basal, preduplicative chordate amphioxus. Surprisingly, however, the origin of the amphioxus cluster is independent of those in vertebrates and drosophilids. Investigation of genomic organization of Iroquois genes in other 17 metazoan genomes revealed a fourth independent 3-gene cluster organization in polychaetes, as well as additional 2- and 4-gene clusters in other clades, in one of the most striking examples of convergence in genomic organization described so far. The recurrent independent evolution of Iroquois clusters suggests a functional importance of this organization for these genes, perhaps related to the sharing of regulatory elements. Consistent with this, comparative analysis of genomic regions flanking the 3 amphioxus Irx genes revealed several blocks of sequences, conserved for at least 100 Myr. Finally, we discuss the possible causes and implications of the convergent evolution of this genomic and regulatory organization throughout metazoans.     

Loss and gain of domains during evolution of cut superclass homeobox genes

The cut superclass of homeobox genes has been divided into three classes: CUX, ONECUT and SATB. Given the various completed genomes, we have now made a comprehensive survey. We find that there are only two cut domain containing genes in Drosophila, one CUX and one ONECUT type. Caenorhabditis elegans has undergone an expansion of the ONECUT subclass genes and has a gene cluster with three ONECUT class genes, one of which has lost the cut domain. Two of these genes contain a conserved sequence motif, termed OCAM, which also occurs in another gene in C. elegans this motif seems to be nematode specific. A recently uncovered C. elegans CUX gene has sequence conservation in its amino-terminus with vertebrate CUX proteins. Further, the 5' end of this gene containing the conserved region can undergo alternative splicing to give rise to a protein with a different carboxy-terminus lacking the cut- and homeodomain. This protein is conserved in its entirety with vertebrate genes termed CASP--which are also alternative splice products of the CUX genes--and with plant and fungal genes. The highly divergent SATB genes share a conserved amino terminal domain, COMPASS, with the Drosophila defective proventriculus gene and a C. elegans ORF. These two "COMPASS" family genes encode two highly divergent homeodomains, may be homologues of the SATB genes and thus probably belong to the cut superclass, too.     

Vertebrate homeobox genes

Recent highlights in vertebrate homeobox gene research include the discovery of new genes with novel expression patterns, observations that peptide growth factors and retinoic acid influence homeobox gene expression, and the generation of mutant phenotypes of embryos homozygous for null mutations. These combined studies reinforce the idea that homeobox genes function near the top of the gene hierarchies controlling vertebrate embryogenesis.     

Vertebrate homeobox genes

In the former part of the review the principal available data aboutHox genes, their molecular organisation and their expression in vertebrate embryos, with particular emphasis for mammals, are briefly summarized.In the latter part we analysed the expression of four mouse homeobox genes related to twoDrosophila genes expressed in the developing head of the fly: Emx1 and Emx2, related toems, and Otx1 and Otx2, related tootd.     

An orphan PRD class homeobox gene expressed in mouse brain and limb development

We report the cDNA sequence and expression of a mouse homeobox gene, Dmbx1, from the PRD class and comparison to its human orthologue. The gene defines a new homeobox gene family, Dmbx, phylogenetically distinct from the Ptx, Alx, Prx Otx, Gsc, Otp and Pax gene families. The Dmbx1 gene is expressed in the developing mouse diencephalon, midbrain and hindbrain, and has dynamic expression during forelimb and hindlimb development. Unusually for homeobox genes, there is no orthologue in the Drosophila or Caenorhabditis genomes; we argue this reflects secondary loss.     

Gradient fields and homeobox genes.

We review here old experiments that defined the existence of morphogenetic gradient fields in vertebrate embryos. The rather abstract idea of cell fields of organ-forming potential has become less popular among modern developmental and molecular biologists. Results obtained with antibodies directed against homeodomain proteins suggest that gradient fields may indeed be visualized at the level of individual regulatory molecules in vertebrate embryos.     

Cloning of rat homeobox genes

We report the isolation of nine rat cognates of mouse homeoboxes within the fourHox gene clusters and a rat homologue of mouseIPF1 homeobox,RHbox# 13A. The sequences of nine cloned homeoboxes are highly similar to those of the mouse and human homeoboxes in the Hox clusters. The restriction enzyme sites and map distances between each of the homeoboxes on the rat genome are nearly identical to those of mouse and human. Thus, we conclude that the isolated homeoboxes are the rat homologues of mouse homeoboxes within the four Hox clusters. A novel homeoboxRHbox# 13A is different from theDrosophila Antennapedia (Antp) sequence but is highly similar to theXlHbox8 (Xenopus laevis) andHtrA2 (Helobdella triserialis) homeoboxes. Forty-two amino acids of the last two-thirds of theRHbox# 13A, XlHbox8, and mouseIPF1 homeodomains completely matched. In addition, these four homeodomains contain a unique His residue in the recognition helix of a helix-turn-helix DNA-binding motif. This His residue is not found in any of the previously published mammalian homeodomain sequences except mouseIPF1.     

Ancient complexity of the non-Hox ANTP gene complement in the anthozoan Nematostella vectensis: implications for the evolution of the ANTP superclass

The origin and evolution of ANTP superclass genes has raised controversial discussions. While recent evidence suggests that a true Hox cluster emerged after the cnidarian bilaterian split, the origin of the ANTP superclass as a whole remains unclear. Based on analyses of bilaterian genomes, it seems very likely that clustering has once been a characteristic of all ANTP homeobox genes and that their ancestors have emerged through several series of cis-duplications from the same genomic region. Since the diploblastic Cnidaria possess orthologs of some non-Hox ANTP genes, at least some steps of the expansion of this hypothetical homeobox gene array must have occurred in the last common ancestor of both lineages--but it is unknown to what extent. By screening the unassembled Nematostella genome, we have identified unambiguous orthologs to almost all non-Hox ANTP genes which are present in Bilateria--with the exception of En, Tlx and (possibly) Vax. Furthermore, Nematostella possesses ANTP genes that are missing in some bilaterian lineages, like the rough gene or NK7. In addition, several ANTP homeobox gene families have been independently duplicated in Nematostella. We conclude that the last cnidarian/bilaterian ancestor already harboured the almost full complement of non-Hox ANTP genes before the Hox system evolved.     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

The genesis and evolution of homeobox gene clusters

Once called the 'Rosetta stone' of developmental biology, the homeobox continues to fascinate both evolutionary and developmental biologists. The birth of the homeotic, or Hox, gene cluster, and its subsequent evolution, has been crucial in mediating the major transitions in metazoan body plan. Comparative genomics studies indicate that the more recently discovered ParaHox and NK clusters were linked to the Hox cluster early in evolution, and that together they constituted a 'megacluster' of homeobox genes that conspicuously contributed to body-plan evolution.