Organization of the Hox gene cluster of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: a split of the Hox cluster in a non-<Emphasis Type="Italic">Drosophila</Emphasis> insect

A bacterial artificial chromosome (BAC) contig was constructed by chromosome walking, starting from the Hox genes of the silkworm, Bombyx mori. Bombyx orthologues of the labial (lab) and zerknült (zen) genes were newly identified. The size of the BAC contig containing the Hox gene cluster—except the lab and Hox 2 genes—was estimated to be more than 2 Mb. The Bombyx Hox cluster was mapped to linkage group (LG) 6. The lab gene was mapped on the same LG, but far apart from the cluster. Fluorescence in situ hybridization analysis confirmed that the major Hox gene cluster and lab were at different locations on the same chromosome in B. mori.Edited by M. Akam     

Evolution of echinoderms may not have required modification of the ancestral deuterostome HOX gene cluster: first report of PG4 and PG5 Hox orthologues in echinoderms

Is the extreme derivation of the echinoderm body plan reflected in a derived echinoderm Hox genotype? Building on previous work, we exploited the sequence conservation of the homeobox to isolate putative orthologues of several Hox genes from two asteroid echinoderms. The 5-peptide motif (LPNTK) diagnostic of PG4 Hox genes was identified immediately downstream of one of the partial homeodomains from Patiriella exigua. This constitutes the first unequivocal report of a PG4 Hox gene orthologue from an echinoderm. Subsequent screenings identified genes of both PG4 and PG4/5 in Asterias rubens. Although in echinoids only a single gene (PG4/5) occupies these two contiguous cluster positions, we conclude that the ancestral echinoderm must have had the complete deuterostome suite of medial Hox genes, including orthologues of both PG4 and PG4/5 (= PG5). The reported absence of PG4 in the HOX cluster of echinoids is therefore a derived state, and the ancestral echinoderm probably had a HOX cluster not dissimilar to that of other deuterostomes. Modification of the ancestral deuterostome Hox genotype may not have been required for evolution of the highly derived echinoderm body plan.     

Evolution of <Emphasis Type="Italic">Hox3</Emphasis> and <Emphasis Type="Italic">ftz</Emphasis> in arthropods: insights from the crustacean <Emphasis Type="Italic">Daphnia pulex</Emphasis>

The Drosophila melanogaster genes zerknüllt (zen) and fushi tarazu (ftz) are members of the Hox gene family whose roles have changed significantly in the insect lineage and thus provide an opportunity to study the mechanisms underlying the functional evolution of Hox proteins. We have studied the expression of orthologs of zen (DpuHox3) and ftz (Dpuftz) in the crustacean Daphnia pulex (Branchiopoda), both of which show a dynamic expression pattern. DpuHox3 is expressed in a complex pattern in early embryogenesis, with the most anterior boundary of expression lying at the anterior limit of the second antennal segment as well as a ring of expression around the embryo. In later embryos, DpuHox3 expression is restricted to the mesoderm of mandibular limb buds. Dpuftz is first expressed in a ring around the embryo following the posterior limit of the mandibular segment. Later, Dpuftz is restricted to the posterior part of the mandibular segment. This is the first report of expression of a Hox3 ortholog in a crustacean, and together with Dpuftz data, the results presented here show that Hox3 and ftz have retained a Hox-like expression pattern in crustaceans. This is in accordance with the proposed model of Hox3 and ftz evolution in arthropods and allows a more precise pinpointing of the loss of ftz “Hox-like behaviour”: in the lineage between the Branchiopoda and the basal insect Thysanura.     

Hox gene expression in larval development of the polychaetes Nereis virens and Platynereis dumerilii (Annelida, Lophotrochozoa)

The bilaterian animals are divided into three great branches: the Deuterostomia, Ecdysozoa, and Lophotrochozoa. The evolution of developmental mechanisms is less studied in the Lophotrochozoa than in the other two clades. We have studied the expression of Hox genes during larval development of two lophotrochozoans, the polychaete annelids Nereis virens and Platynereis dumerilii. As reported previously, the Hox cluster of N. virens consists of at least 11 genes (de Rosa R, Grenier JK, Andreeva T, Cook CE, Adoutte A, Akam M, Carroll SB, Balavoine G, Nature, 399:772–776, 1999; Andreeva TF, Cook C, Korchagina NM, Akam M, Dondua AK, Ontogenez 32:225–233, 2001); we have also cloned nine Hox genes of P. dumerilii. Hox genes are mainly expressed in the descendants of the 2d blastomere, which form the integument of segments, ventral neural ganglia, pre-pygidial growth zone, and the pygidial lobe. Patterns of expression are similar for orthologous genes of both nereids. In Nereis, Hox2, and Hox3 are activated before the blastopore closure, while Hox1 and Hox4 are activated just after this. Hox5 and Post2 are first active during the metatrochophore stage, and Hox7, Lox4, and Lox2 at the late nectochaete stage only. During larval stages, Hox genes are expressed in staggered domains in the developing segments and pygidial lobe. The pattern of expression of Hox cluster genes suggests their involvement in the vectorial regionalization of the larval body along the antero-posterior axis. Hox gene expression in nereids conforms to the canonical patterns postulated for the two other evolutionary branches of the Bilateria, the Ecdysozoa and the Deuterostomia, thus supporting the evolutionary conservatism of the function of Hox genes in development. Milana Kulakova, Nadezhda Bakalenko and Elena Novikova contributed equally to this work.     

Isolation and expression analysis of a poriferan <Emphasis Type="Italic">Antp</Emphasis>-class <Emphasis Type="Italic">Bar-/Bsh-</Emphasis>like homeobox gene

A novel non-Hox Antp-class gene (BarBsh-Hb) was isolated from the marine sponge Halichondria sp. This gene shares high sequence identity with eumetazoan genes from the Bsh and Bar gene families and can be distinguished from other non-Hox Antp-class genes by diagnostic residues. We also present an alignment of all known (full-length) poriferan non-Hox Antp-class genes. Maximum likelihood methods were employed to estimate phylogenetic relationships among non-Hox genes and BarBsh-Hb. We employed RT-PCR techniques to look at expression across different developmental stages (larval to rhagon). BarBsh-Hb product was present in newly released larvae, but expression was not detected 8–16 h post-release. Expression of BarBsh-Hb was detected in later-stage (>16 h post-release), free-swimming larvae until they settled and attached to the substratum, after which expression was down-regulated. In a separate set of experiments, low levels of expression were observed in normal adult tissue and disaggregated adult tissue, but BarBsh-Hb expression increased during tissue re-aggregation. These data increase the number of non-Hox homeobox genes identified in sponges and provide evidence of regulation of this non-Hox gene during sponge development. While the Bar and Bsh genes play important roles in the development of nervous tissue—especially visual systems—in metazoans, the specific role(s) BarBsh-Hb play(s) in sponge development is unclear and deserves greater attention.Edited by C. Desplan     

Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development

Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region. Egr2, which is used in this study as an early developmental marker of rhombomeres 3 and 5, is expressed in two distinct bands with a location and spacing typical for these two rhombomeres in other species. Within the pharyngeal arches, hoxa2a is expressed at higher levels in the second pharyngeal arch, while hoxa2b is more strongly expressed in the posterior arches. Further, hoxa2b expression within the arches becomes undetectable at 60hpf, while hoxa2a expression is maintained at least up until the beginning of chondrogenesis. Comparison of the striped bass HoxA cluster paralog group 2 (PG2) genes to their orthologs and trans-orthologs shows that the striped bass hoxa2a gene expression pattern is similar to the overall expression pattern described for the hoxa2 genes in the lobe-finned fish lineage and for the hoxa2b gene from zebrafish. It is notable that the pharyngeal arch expression pattern of the striped bass hoxa2a gene is more divergent from its sister paralog, hoxa2b, than from the zebrafish hoxa2b gene. Overall, our results suggest that differences in the Hox PG2 gene complement of striped bass and zebrafish affects both their rhombomeric and pharyngeal arch expression patterns and may account for the similarities in pharyngeal arch expression between striped bass hoxa2a and zebrafish hoxa2b.     

Hox Genes from the Tapeworm Taenia asiatica (Platyhelminthes: Cestoda)

Hox genes are important in forming the anterior-posterior body axis pattern in the early developmental stage of animals. The conserved nature of the genomic organization of Hox genes is well known in diverse metazoans. To understand the Hox gene architecture in human-infecting Taenia tapeworms, we conducted a genomic survey of the Hox gene using degenerative polymerase chain reaction primers in Taenia asiatica. Six Hox gene orthologs from 276 clones were identified. Comparative analysis revealed that T. asiatica has six Hox orthologs, including two lab/Hox1, two Hox3, one Dfd/Hox4, and one Lox2/Lox4. The results suggest that Taenia Hox genes may have undergone independent gene duplication in two Hox paralogs. The failure to detect Post1/2 orthologs in T. asiatica may suggest that sequence divergence or the secondary loss of the posterior genes has occurred in the lineage leading to the cestode and trematode.     

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.     

The<Emphasis Type="Italic"> Trox-2</Emphasis> Hox/ParaHox gene of<Emphasis Type="Italic"> Trichoplax</Emphasis> (Placozoa) marks an epithelial boundary

Hox and ParaHox genes are implicated in axial patterning of cnidarians and bilaterians, and are thought to have originated by tandem duplication of a single ProtoHox gene followed by duplication of the resultant gene cluster. It is unclear what the ancestral role of Hox/ParaHox genes was before the divergence of Cnidaria and Bilateria, or what roles the postulated ProtoHox gene(s) played. Here we describe the full coding region, spatial expression and function of Trox-2, the single Hox/ParaHox-type gene identified in Trichoplax adhaerens (phylum Placozoa) and either a candidate ProtoHox or a ParaHox gene. Trox-2 is expressed in a ring around the periphery of Trichoplax, in small cells located between the outer margins of the upper and lower epithelial cell layers. Inhibition of Trox-2 function, either by uptake of morpholino antisense oligonucleotides or by RNA interference, causes complete cessation of growth and binary fission. We speculate that Trox-2 functions within a hitherto unrecognized population of possibly multipotential peripheral stem cells that contribute to differentiated cells at the epithelial boundary of Trichoplax.Edited by D. Tautz     

Hox genes in the echiuroid Urechis unicinctus

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.     

Isolation of Hox and Parahox genes in the hemichordate Ptychodera flava and the evolution of deuterostome Hox genes

Because of their importance for proper development of the bilaterian embryo, Hox genes have taken center stage for investigations into the evolution of bilaterian metazoans. Taxonomic surveys of major protostome taxa have shown that Hox genes are also excellent phylogenetic markers, as specific Hox genes are restricted to one of the two great protostome clades, the Lophotrochozoa or the Ecdysozoa, and thus support the phylogenetic relationships as originally deduced by 18S rDNA studies. Deuterostomes are the third major group of bilaterians and consist of three major phyla, the echinoderms, the hemichordates, and the chordates. Most morphological studies have supported Hemichordata+Chordata, whereas molecular studies support Echinodermata+Hemichordata, a clade known as Ambulacraria. To test these competing hypotheses, complete or near complete cDNAs of eight Hox genes and four Parahox genes were isolated from the enteropneust hemichordate Ptychodera flava. Only one copy of each Hox gene was isolated suggesting that the Hox genes of P. flava are arranged in a single cluster. Of particular importance is the isolation of three posterior or Abd-B Hox genes; these genes are only shared with echinoderms, and thus support the monophyly of Ambulacraria.