Evolution of the echinoderm Hox gene cluster

SUMMARY Extant echinoderms are members of an ancient and highly derived deuterostome phylum. The composition and arrangement of their Hox gene clusters are consequently of interest not only from the perspective of evolution of development, but also in terms of metazoan phylogeny and body plan evolution. Over the last decade numerous workers have reported partial Hox gene sequences from a variety of echinoderms. In this paper we used a combined methods approach to analyze phylogenetic relationships between 68 echinoderm Hox homeodomain fragments, from species of five extant classes—two asteroids, one crinoid, one ophiuroid, one holothuroid, and three echinoids. This analysis strengthens Mito and Endo's (2000) proposition that the ancestral echinoderm's Hox gene cluster contained at least eleven genes, including at least four posterior paralogous group genes. However, representatives of all paralogous groups are not known from all echinoderm classes. In particular, these data suggest that echinoids may have lost a posterior group Hox gene subsequent to the divergence of the echinoderm classes. Evolution of the highly derived echinoderm body plan may have been accompanied by class-specific duplication, diversification and loss of Hox genes.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

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.     

Genomic organization of Hox and ParaHox clusters in the echinoderm,Acanthaster planci

The organization of echinoderm Hox clusters is of interest due to the role that Hox genes play in deuterostome development and body plan organization, and the unique gene order of the Hox complex in the sea urchin Strongylocentrotus purpuratus, which has been linked to the unique development of the axial region. Here, it has been reported that the Hox and ParaHox clusters of Acanthaster planci, a corallivorous starfish found in the Pacific and Indian oceans, generally resembles the chordate and hemichordate clusters. The A. planci Hox cluster shared with sea urchins the loss of one of the medial Hox genes, even‐skipped (Evx) at the anterior of the cluster, as well as organization of the posterior Hox genes. genesis 52:952–958, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Identification of Hox Gene Sequences in the Sea Cucumber Holothuria glaberrima Selenka (Holothuroidea: Echinodermata)

The Echinodermata is a unique animal group forming an early branch in the deuterostomes phylogenetic tree. In echinoids and asteroids a single Hox cluster with nine cognates of the vertebrate Hox paralogous groups has been reported, but no data are available from other echinoderm classes. We report here nine Hox-type sequences from the sea cucumber Holothuria glaberrima, a member of the class Holothuroidea. Partial homeodomain sequences were amplified by polymerase chain reaction from genomic DNA and from a regenerating gastrointestinal tract complementary DNA library. Sequence analyses suggest that the holothuroid cluster has at least three genes of the anterior, one of the medial, and five of the posterior groups. This is the first evidence of five posterior sequences in echinoderms. Received July 19, 1999; accepted October 18, 1999.     

Two more Posterior Hox genes and Hox cluster dispersal in echinoderms

Background

Hox genes are key elements in patterning animal development. They are renowned for their, often, clustered organisation in the genome, with supposed mechanistic links between the organisation of the genes and their expression. The widespread distribution and comparable functions of Hox genes across the animals has led to them being a major study system for comparing the molecular bases for construction and divergence of animal morphologies. Echinoderms (including sea urchins, sea stars, sea cucumbers, feather stars and brittle stars) possess one of the most unusual body plans in the animal kingdom with pronounced pentameral symmetry in the adults. Consequently, much interest has focused on their development, evolution and the role of the Hox genes in these processes. In this context, the organisation of echinoderm Hox gene clusters is distinctive. Within the classificatory system of Duboule, echinoderms constitute one of the clearest examples of Disorganized (D) clusters (i.e. intact clusters but with a gene order or orientation rearranged relative to the ancestral state).

Results

Here we describe two Hox genes (Hox11/13d and e) that have been overlooked in most previous work and have not been considered in reconstructions of echinoderm Hox complements and cluster organisation. The two genes are related to Posterior Hox genes and are present in all classes of echinoderm. Importantly, they do not reside in the Hox cluster of any species for which genomic linkage data is available.

Conclusion

Incorporating the two neglected Posterior Hox genes into assessments of echinoderm Hox gene complements and organisation shows that these animals in fact have Split (S) Hox clusters rather than simply Disorganized (D) clusters within the Duboule classification scheme. This then has implications for how these genes are likely regulated, with them no longer covered by any potential long-range Hox cluster-wide, or multigenic sub-cluster, regulatory mechanisms.

     

Anterior boundaries of Hox gene expression in mesoderm-derived structures correlate with the linear gene order along the chromosome

The developmental expression patterns of four genes, Hox 1.1, Hox 1.2, Hox 1.3 and Hox 3.1, were examined by in situ hybridization to serial embryonic sections. The three genes of the Hox 1 cluster, used in this study, map to adjacent positions along chromosome 6, whereas the Hox 3.1 gene maps to the Hox 3 cluster on chromosome 15. The anterior expression limits in segmented mesoderm varied among the four genes examined. Interestingly, a linear correlation exists between the position of the gene along the chromosome and the extent of anterior expression. Genes that are expressed more posterior are also more restricted in their expression in other mesoderm-derived tissues. The order of expression anterior to posterior was determined as: Hox 1.3, Hox 1.2, Hox 1.1 and Hox 3.1. Similarly, genes of the Drosophila Antennapedia and Bithorax complex specifying segment identity also exhibit anterior expression boundaries that correlate with gene position. The data suggest that Hox genes may specify positional information along the anterior-posterior axis during the formation of the body plan.     

Hox clusters and bilaterian phylogeny

A large Hox cluster comprising at least seven genes has evolved by gene duplications in the ancestors of bilaterians. It probably emerged from a mini-cluster of three or four genes that was present before the divergence of cnidarians and bilaterians. The comparison of Hox structural data in bilaterian phyla shows that the genes of the anterior part of the cluster have been more conserved than those of the posterior part. Some specific signature sequences, present in the form of signature residues within the homeodomain or conserved peptides outside the homeodomain, constitute phylogenetic evidence for the monophyly of protostomes and their division into ecdysozoans and lophotrochozoans. These conserved motifs may provide decisive arguments for the phylogenetic position of some enigmatic phyla.     

Hox genes from the earthworm Perionyx excavatus

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.     

Hox and paraHox genes from the anthozoan Parazoanthus parasiticus

We surveyed the genome of the Caribbean zoanthid Parazoanthus parasiticus for Hox and paraHox genes, and examined gene expression patterns for sequences we uncovered. Two Hox genes and three paraHox genes were identified in our surveys. The Hox genes belong to anterior and posterior classes. In phylogenetic analyses, the anterior Hox sequence formed an anthozoan-specific cluster that appears to be a second class of cnidarian anterior Hox gene. The presence of an anterior Gsx-like paraHox gene supports the hypothesis that duplication of a protoHox gene family preceded the divergence of the Cnidaria and bilaterians. The presence of two Mox class paraHox genes in P. parasiticus deserves further attention. Expression analysis using RT-PCR, indicated that one Mox gene and the anterior paraHox gene are not expressed in adult tissue, whereas the other three sequences are expressed in both dividing and unitary polyps. Dividing polyps showed slightly lower Ppox1 (i.e., Mox) expression levels. Our data add to the number of published anthozoan sequences, and provide additional detail concerning the evolutionary significance of cnidarian Hox and paraHox genes.     

Genomic and gene regulatory signatures of cryptozoic adaptation: Loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum

Background

Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster.

Results

We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo.

Conclusion

The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries.     

Homeobox Genes Expressed During Echinoderm Arm Regeneration

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.     

Colinear and segmental expression of amphioxus Hox genes.

The cephalochordate amphioxus has a single Hox gene cluster. Here we describe the genomic organization of four adjacent amphioxus genes, AmphiHox-1 to AmphiHox-4, together with analysis of their spatiotemporal expression patterns. We demonstrate that these genes obey temporal colinearity and that three of the genes also obey spatial colinearity in the developing neural tube. AmphiHox-1, AmphiHox-3, and AmphiHox-4 show segmental modulation of their expression levels, a two-segment phasing of spatial colinearity, and, at least for AmphiHox-4, asymmetrical expression. AmphiHox-2 is unlike other amphioxus Hox genes: it does not obey spatial colinearity and it has no positional expression in the neural tube. AmphiHox-2 is expressed in the preoral pit of larvae, from which the homologue of the anterior pituitary develops. We suggest that the ancestral role of chordate Hox genes was primarily in the neural tube and that chordate Hox genes can functionally diverge in a manner analogous to that of Drosophila ftz or zen.     

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.     

Hox and ParaHox genes in Nemertodermatida, a basal bilaterian clade

Molecular evidence suggests that Acoelomorpha, a proposed phylum composed of acoel and Nemertodermatida flatworms, are the most basal bilaterian animals. Hox and ParaHox gene complements characterised so far in acoels consist of a small set of genes, comprising representatives of anterior, central and posterior genes, altogether Hox and ParaHox, but no PG3-Xlox representatives have been reported. It has been proposed that this might be the ancestral Hox repertoire in basal bilaterians. However, no studies of the other members of the group, the Nemertodermatida, have been done. In order to get a more complete picture of the basal bilaterian Hox and ParaHox complement, we have analysed the Hox/ParaHox complement of the nemertodermatid Nemertoderma westbladi. We have found representatives of two central and one posterior Hox genes, as well as an Xlox and a Caudal ParaHox gene. From our data we conclude that a PG3-Xlox gene was present in the ancestor of bilaterians. These findings support the speculation that basal bilaterians already had the beginnings of the extended central Hox set, driving back gene duplications in the central part of the Hox cluster deeper in phylogeny than previously suggested.     

The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14

SUMMARY The amphioxus ( Branchiostoma floridae ) Hox cluster is a model for the ancestral vertebrate cluster, prior to the hypothesized genome-wide duplications that may have facilitated the evolution of the vertebrate body plan. Here we describe the posterior (5') genes of the amphioxus cluster, and report the isolation of four new homeobox genes. Vertebrates possess 13 types of Hox gene (paralogy groups), but we show that amphioxus possesses more than 13 Hox genes. Amphioxus is now the first animal in which a Hox14 gene has been found. Our mapping and phylogenetic analysis of amphioxus "Posterior Class" Hox genes reveals that these genes are evolving at a faster rate in deuterostomes than in protostomes, a phenomenon we term Posterior Flexibility.     

Hox and paraHox genes in bivalve molluscs

In this study, we sought the presence and analysed the sequences of the Hox and ParaHox genes in bivalve molluscs. The clustered Hox genes play a central role in anterior-posterior axial patterning in bilaterian metazoa, whereas the ParaHox gene cluster is a paralogue (evolutionary sister) of the Hox cluster.Using polymerase chain reaction (PCR)-based approaches, we isolated nine different sequences in five species belonging to three of the main bivalve subclasses: Ensis ensis and Tapes philippinarum (Heterodonta), Pecten maximus and Mytilus galloprovincialis (Pteriomorphia), and Yoldia eightsi (Protobranchia). Comparison with the Hox and ParaHox genes of other bilaterians, particularly lophotrochozoans, allowed us to attribute six of these sequences to the Hox gene cluster (one to paralog group [PG] 3 class, and five to the central class), two to the ParaHox cluster and one to the Gbx gene family.The results of our investigation seem to indicate that homeotic Hox and ParaHox gene clusters are homogeneous for both presence and characteristics in molluscs.