Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments

The pycnogonids (or sea spiders) are an enigmatic group of arthropods, classified in recent phylogenies as a sister-group of either euchelicerates (horseshoe crabs and arachnids), or all other extant arthropods. Because of their bizarre morpho-anatomy, homologies with other arthropod taxa have been difficult to assess. We review the main morphology-based hypotheses of correspondence between anterior segments of pycnogonids, arachnids and mandibulates. In an attempt to provide new relevant data to these controversial issues, we performed a PCR survey of Hox genes in two pycnogonid species, Endeis spinosa and Nymphon gracile, from which we could recover nine and six Hox genes, respectively. Phylogenetic analyses allowed to identify their orthology relationships. The Deformed gene from E. spinosa and the abdominal-A gene from N. gracile exhibit unusual sequence divergence in their homeodomains, which, in the latter case, may be correlated with the extreme reduction of the posterior region in pycnogonids. Expression patterns of two Hox genes (labial and Deformed) in the E. spinosa protonymphon larva are discussed. The anterior boundaries of their expression domains favour homology between sea spider chelifores, euchelicerates chelicerae and mandibulate (first) antennae, in contradistinction with previously proposed alternative schemes such as the protocerebral identity of sea spider chelifores or the absence of a deutocerebrum in chelicerates. In addition, while anatomical and embryological evidences suggest the possibility that the ovigers of sea spiders could be a duplicated pair of pedipalps, the Hox data support them as modified anterior walking legs, consistent with the classical views.Supplementary material is available for this article at and is accessible for authorized users.Guest editors Jean Deutsch and Gerhard Scholtz     

Tribolium Hox genes repress antennal development in the gnathos and trunk

Evidence from Drosophila suggests that Hox genes not only specify regional identity, but have the additional function of repressing antennal development within their normal domains. This is dramatically demonstrated by a series of Hox mutants in the red flour beetle, Tribolium castaneum, and is likely an ancient function of Hox genes in insects.     

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.     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

Isolation of Hox genes from the scyphozoan Cassiopeia xamachana: implications for the early evolution of Hox genes.

The isolation of Hox genes from two cnidarian groups, the Hydrozoa and Anthozoa, has sparked hypotheses on the early evolution of Hox genes and a conserved role for these genes for defining a main body axis in all metazoan animals. We have isolated the first five Hox genes, Scox-1 to Scox-5, from the third cnidarian class, the Scyphozoa. For all but one gene, we report full-length homeobox plus flanking sequences. Four of the five genes show close relationship to previously reported Cnox-1 genes from Hydrozoa and Anthozoa. One gene, Scox-2, is an unambiguous homologue of Cnox-2 genes known from Hydrozoa, Anthozoa, and also Placozoa. Based on sequence similarity and phylogenetic analyses of the homeobox and homeodomain sequences of known Hox genes from cnidarians, we suggest the presence of at least five distinct Hox gene families in this phylum, and conclude that the last common ancestor of the Recent cnidarian classes likely possessed a set of Hox genes representing three different families, the Cnox-1, Cnox-2, and Cnox-5 families. The data presented are consistent with the idea that multiple duplication events of genes have occurred within one family at the expense of conservation of the original set of genes, which represent the three ancestral Hox gene families.     

Current problems with the zootype and the early evolution of Hox genes

"Hox cluster type" genes have sparked intriguing attempts to unite all metazoan animals by a shared pattern of expression and genomic organization of a specific set of regulatory genes. The basic idea, the zootype concept, claims the conservation of a specific set of "Hox cluster type genes" in all metazoan animals, i.e., in the basal diploblasts as well as in the derived triploblastic animals. Depending on the data used and the type of analysis performed, different opposing views have been taken on this idea. We review here the sum of data currently available in a total evidence analysis, which includes morphological and the most recent molecular data. This analysis highlights several problems with the idea of a simple "Hox cluster type" synapomorphy between the diploblastic and triploblastic animals and suggests that the "zootype differentiation" of the Hox cluster most likely is an invention of the triploblasts. The view presented is compatible with the idea that early Hox gene evolution started with a single proto-Hox (possibly a paraHox) gene. J. Exp. Zool. (Mol. Dev. Evol.) 291:169-174, 2001.     

Hox and ParaHox genes in evolution, development and genomics

The discovery of the homeobox motif and its presence in each gene of the Hox clusters revolutionized the fields of developmental biology and evolutionary developmental biology (1, 2),providing a rapid entrance into investigating the mechanisms of development of almost any animal taxon as well as dramatically altering conceptions on the extent of genetic conservation across the animal kingdom.     

Regressive evolution of the arthropod tritocerebral segment linked to functional divergence of the Hox gene labial

The intercalary segment is a limbless version of the tritocerebral segment and is present in the head of all insects, whereas other extant arthropods have retained limbs on their tritocerebral segment (e.g. the pedipalp limbs in spiders). The evolutionary origin of limb loss on the intercalary segment has puzzled zoologists for over a century. Here we show that an intercalary segment-like phenotype can be created in spiders by interfering with the function of the Hox gene labial. This links the origin of the intercalary segment to a functional change in labial. We show that in the spider Parasteatoda tepidariorum the labial gene has two functions: one function in head tissue maintenance that is conserved between spiders and insects, and a second function in pedipalp limb promotion and specification, which is only present in spiders. These results imply that labial was originally crucial for limb formation on the tritocerebral segment, but that it has lost this particular subfunction in the insect ancestor, resulting in limb loss on the intercalary segment. Such loss of a subfunction is a way to avoid adverse pleiotropic effects normally associated with mutations in developmental genes, and may thus be a common mechanism to accelerate regressive evolution.     

Hox genes from the Polystomatidae (Platyhelminthes, Monogenea)

Hox genes form a multigenic family that play a fundamental role during the early stages of development. They are organised in a single cluster and share a 60 amino acid conserved sequence that corresponds to the DNA binding domain, i.e. the homeodomain. Sequence conservation in this region has allowed investigators to explore Hox diversity in the metazoan lineages. Within parasitic flatworms only homeobox sequences of parasite species from the Cestoda and Digenea have been reported. In the present study we surveyed species of the Polyopisthocotylea (Monogenea) in order to clarify Hox identification and diversification processes in the neodermatan lineage. From cloning of degenerative PCR products of the central region of the homeobox, we report one ParaHox and 25 new Hox sequences from 10 species of the Polystomatidae and one species of the Diclidophoridae, which extend Hox gene diversity from 46 to 72 within Neodermata. Hox sequences from the Polyopisthocotylea were annotated and classified from sequence alignments and Bayesian inferences of 178 Hox, ParaHox and related gene families recovered from all available parasitic platyhelminths and other bilaterian taxa. Our results are discussed in the light of the recent Hox evolutionary schemes. They may provide new perspectives to study the transition from turbellarians to parasitic flatworms with complex life-cycles and outline the first steps for evolutionary developmental biological approaches within platyhelminth parasites.     

Selection on coding regions determined Hox7 genes evolution

The important role of Hox genes in determining the regionalization of the body plan of the vertebrates makes them invaluable candidates for evolutionary analyses regarding functional and morphological innovation. Gene duplication and gene loss led to a variable number of Hox genes in different vertebrate lineages. The evolutionary forces determining the conservation or loss of Hox genes are poorly understood. In this study, we show that variable selective pressures acted on Hox7 genes in different evolutionary lineages, with episodes of positive selection occurring after gene duplications. Tests for functional divergence in paralogs detected significant differentiation in a region known to modulate HOX7 protein activity. Our results show that both positive and negative selection on coding regions are influencing Hox7 genes evolution.     

Hox genes and the phylogeny of the arthropods

The arthropods are the most speciose, and among the most morphologically diverse, of the animal phyla. Their evolution has been the subject of intense research for well over a century, yet the relationships among the four extant arthropod subphyla - chelicerates, crustaceans, hexapods, and myriapods - are still not fully resolved. Morphological taxonomies have often placed hexapods and myriapods together (the Atelocerata) [1, 2], but recent molecular studies have generally supported a hexapod/crustacean clade [2-9]. A cluster of regulatory genes, the Hox genes, control segment identity in arthropods, and comparisons of the sequences and functions of Hox genes can reveal evolutionary relationships [10]. We used Hox gene sequences from a range of arthropod taxa, including new data from a basal hexapod and a myriapod, to estimate a phylogeny of the arthropods. Our data support the hypothesis that insects and crustaceans form a single clade within the arthropods to the exclusion of myriapods. They also suggest that myriapods are more closely allied to the chelicerates than to this insect/crustacean clade.     

Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.     

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.      

Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio)

Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.     

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.