Sipunculan ParaHox genes

SUMMARY Our perspective on the origin and evolution of the Hox gene cluster changed with the discovery of the ParaHox gene cluster in amphioxus (Cephalochordata; Branchiostoma floridae ) ( Brooke et al. 1998 ). The ParaHox gene cluster contains three homeobox genes (Gsx, Xlox, Cdx) and is deduced to be a paralogue (evolutionary sister) of the Hox gene cluster. If this deduction is correct, animals with Hox genes should also possess ParaHox genes. Paradoxically, however, only deuterostome animals have thus far been shown to contain all three ParaHox genes. Here we report the cloning of all three ParaHox genes from each of two species within the phylum Sipuncula. This is the first demonstration of all three ParaHox genes in the genome of a protostome animal and confirms that the common ancestor of protostomes and deuterostomes possessed all three ParaHox genes. Furthermore, it implies that the ParaHox genes are of sufficient functional importance in both protostomes and deuterostomes that they have all been conserved in both of these bilaterian clades.     

The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.Bum Joon Park and Sung-Jin Cho contributed equally to this work.     

Ancient origins of axial patterning genes: Hox genes and ParaHox genes in the Cnidaria

Among the bilaterally symmetrical, triploblastic animals (the Bilateria), a conserved set of developmental regulatory genes are known to function in patterning the anterior–posterior (AP) axis. This set includes the well-studied Hox cluster genes, and the recently described genes of the ParaHox cluster, which is believed to be the evolutionary sister of the Hox cluster ( Brooke et al. 1998 ). The conserved role of these axial patterning genes in animals as diverse as frogs and flies is believed to reflect an underlying homology (i.e., all bilaterians derive from a common ancestor which possessed an AP axis and the developmental mechanisms responsible for patterning the axis). However, the origin and early evolution of Hox genes and ParaHox genes remain obscure. Repeated attempts have been made to reconstruct the early evolution of Hox genes by analyzing data from the triphoblastic animals, the Bilateria ( Schubert et al. 1993 ; Zhang and Nei 1996 ). A more precise dating of Hox origins has been elusive due to a lack of sufficient information from outgroup taxa such as the phylum Cnidaria (corals, hydras, jellyfishes, and sea anemones). In combination with outgroup taxa, another potential source of information about Hox origins is outgroup genes (e.g., the genes of the ParaHox cluster). In this article, we present cDNA sequences of two Hox-like genes ( anthox2 and anthox6 ) from the sea anemone, Nematostella vectensis. Phylogenetic analysis indicates that anthox2 (=Cnox2) is homologous to the GSX class of ParaHox genes, and anthox6 is homologous to the anterior class of Hox genes. Therefore, the origin of Hox genes and ParaHox genes occurred prior to the evolutionary split between the Cnidaria and the Bilateria and predated the evolution of the anterior–posterior axis of bilaterian animals. Our analysis also suggests that the central Hox class was invented in the bilaterian lineage, subsequent to their split from the Cnidaria.     

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.     

A degenerate ParaHox gene cluster in a degenerate vertebrate

The ParaHox genes consist of 3 homeobox gene families, Gsx, Xlox, and Cdx, all of which have fundamental roles in development. Xlox (known as IPF1 or PDX1 in vertebrates), for example, is crucial for development of the vertebrate pancreas and is also involved in regulation of insulin expression. The invertebrate amphioxus has a gene cluster containing one gene from each of the gene families, whereas in all vertebrates examined to date there are additional copies resultant from ParaHox gene cluster duplications at the base of the vertebrate lineage. Extant vertebrates basal to bony and cartilaginous fish are central to the question of when and how these multiple genes arose in the vertebrate genome. Here, we report the mapping of a ParaHox gene cluster in 2 species of hagfishes. Unexpectedly, these basal vertebrates have lost a functional Xlox gene from this cluster, unlike every other vertebrate examined to date. Furthermore, our phylogenetic analyses suggest that hagfishes may have diverged from the vertebrate lineage before the duplications, which created the multiple ParaHox clusters in jawed vertebrates.     

Ambulacrarian prototypical Hox and ParaHox gene complements of the indirect-developing hemichordate <Emphasis Type="Italic">Balanoglossus simodensis</Emphasis>

The Hox genes and its evolutionary sister, the ParaHox genes, are widely distributed among animals. Although it has been expected that hemichordates and echinoderms have a single set of Hox genes and most likely a single set of ParaHox genes, it is not known whether the ortholog of Hox8 is absent in hemichordates, and in turn, consensus view about Hox/ParaHox gene complements in hemichordates has not been established. In this study, we isolated either complete or nearly complete coding sequences of 12 Hox genes, including the ortholog of the Hox8 that has not been reported in the previous studies, and three ParaHox genes from the recently discovered indirect-developing acorn worm, Balanoglossus simodensis. Our data suggest that the ancestral hemichordate had intact complements of ambulacrarian prototypical Hox and ParaHox genes, consisting of 12 and three members, respectively.     

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.     

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.     

Evolution of the vertebrate ParaHox clusters

The ParaHox cluster contains three Hox-related homeobox genes. The evolution of this sister of the Hox-gene clusters has been studied extensively in metazoans with a focus on its early evolution. Its fate within the vertebrate lineage, and in particular following the teleost-specific genome duplication, however, has not received much attention. Three of the four human ParaHox loci are linked with PDGFR family tyrosine kinases. We demonstrate that these loci arose as duplications in an ancestral vertebrate and trace the subsequent history of gene losses. Surprisingly, teleost fishes have not expanded their ParaHox repertoire following the teleost-specific genome duplication, while duplicates of the associated tyrosine kinases have survived, supporting the hypothesis of a large-scale duplication followed by extensive gene loss.     

Combined-method phylogenetic analysis of Hox and ParaHox genes of the metazoa

The clustered Hox genes show a conserved role in patterning the body axis of bilaterian metazoans. Increasingly, a broader phylogenetic sampling of non-model system organisms is being examined to detect a correlation, if any, between Hox gene evolution, and body plan innovations. To assess how Hox gene expression and function evolve with changing cluster arrangements, we must be able to reliably assign gene orthologies between Hox genes. Recent evidence suggests that a four-gene proto-Hox cluster duplicated to form the precursor of the present cluster and an additional sister-cluster, the ParaHox group. Here, phylogenetic methods are used to determine Hox-gene orthologies and to infer probable clustering events leading to the current bilaterian Hox complement. This analysis supports the ParaHox hypothesis and gives first confirmation that ind (intermediate neuroblasts defective) is an anterior ParaHox ortholog from protostomes. This analysis supports a proto-Hox cluster of four genes in which the central-class member of the ParaHox cluster may have been lost. It is also proposed here that ancestral diploblasts had central-class members of both Hox and ParaHox clusters. Primitive Hox gene ancestors are estimated by phylogenetic methods and found to have no strong affinity to any particular class of extant Hox members.     

Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae)

The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid (RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posterior position. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes. By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster.     

Ciona intestinalis ParaHox genes: evolution of Hox/ParaHox cluster integrity,developmental mode,and temporal colinearity

The Hox gene cluster, and its evolutionary sister the ParaHox gene cluster, pattern the anterior-posterior axis of animals. The spatial and temporal regulation of the genes seems to be intimately linked to the gene order within the clusters. In some animals the tight organisation of the clusters has disintegrated. We note that these animals develop in a derived fashion relative to the norm of their respective lineages. Here we present the genomic organisation of the ParaHox genes of Ciona intestinalis, and note that tight clustering has been lost in evolution. We present a hypothesis that the Hox and ParaHox clusters are constrained as ordered clusters by the mechanisms producing temporal colinearity; when temporal colinearity is no longer needed or used during development, the clusters can fall apart. This disintegration may be mediated by the invasion of transposable elements into the clusters, and subsequent genomic rearrangements.     

Hox, ParaHox, ProtoHox: facts and guesses

The Hox gene cluster has captivated the imagination of evolutionary and developmental biologists worldwide. In this review, the origin of the Hox and ParaHox gene clusters by duplication of a ProtoHox gene cluster, and the changes in their gene numbers in major Metazoan Transitions are reviewed critically. Re-evaluation of existing data and recent findings in Cnidarians, Acoels, and critical stages of vertebrate evolution suggest alternative scenarios for the origin, structure, and changes in Hox gene numbers in relevant events of Metazoan evolution. I discuss opposing views and propose that (i) the ProtoHox cluster had only two genes, and not four as commonly believed: a corollary is that the origin of Bilaterians was coincident with the invention of new Hox and ParaHox gene classes, which may have facilitated such a transition; (ii) the ProtoHox cluster duplication was a cis duplication event, rather than a trans duplication event, as previously suggested, and (iii) the ancestral vertebrate cluster possessed 14 Hox genes, and not the 13 generally assumed. These hypotheses could be verified or refuted in the near future, but they may help critical discussion of the evolution of the Hox/ParaHox family in the metazoan kingdom.     

Do cnidarians have a ParaHox cluster? Analysis of synteny around a Nematostella homeobox gene cluster

SUMMARY The Hox gene cluster is renowned for its role in developmental patterning of embryogenesis along the anterior–posterior axis of bilaterians. Its supposed evolutionary sister or paralog, the ParaHox cluster, is composed of Gsx, Xlox, and Cdx, and also has important roles in anterior–posterior development. There is a debate as to whether the cnidarians, as an outgroup to bilaterians, contain true Hox and ParaHox genes, or instead the Hox‐like gene complement of cnidarians arose from independent duplications to those that generated the genes of the bilaterian Hox and ParaHox clusters. A recent whole genome analysis of the cnidarian Nematostella vectensis found conserved synteny between this cnidarian and vertebrates, including a region of synteny between the putative Hox cluster of N. vectensis and the Hox clusters of vertebrates. No syntenic region was identified around a potential cnidarian ParaHox cluster. Here we use different approaches to identify a genomic region in N. vectensis that is syntenic with the bilaterian ParaHox cluster. This proves that the duplication that gave rise to the Hox and ParaHox regions of bilaterians occurred before the origin of cnidarians, and the cnidarian N. vectensis has bona fide Hox and ParaHox loci.     

Are Hox Genes Ancestrally Involved in Axial Patterning? Evidence from the Hydrozoan Clytia hemisphaerica (Cnidaria)

Background

The early evolution and diversification of Hox-related genes in eumetazoans has been the subject of conflicting hypotheses concerning the evolutionary conservation of their role in axial patterning and the pre-bilaterian origin of the Hox and ParaHox clusters. The diversification of Hox/ParaHox genes clearly predates the origin of bilaterians. However, the existence of a “Hox code” predating the cnidarian-bilaterian ancestor and supporting the deep homology of axes is more controversial. This assumption was mainly based on the interpretation of Hox expression data from the sea anemone, but growing evidence from other cnidarian taxa puts into question this hypothesis.

Methodology/Principal Findings

Hox, ParaHox and Hox-related genes have been investigated here by phylogenetic analysis and in situ hybridisation in Clytia hemisphaerica, an hydrozoan species with medusa and polyp stages alternating in the life cycle. Our phylogenetic analyses do not support an origin of ParaHox and Hox genes by duplication of an ancestral ProtoHox cluster, and reveal a diversification of the cnidarian HOX9-14 genes into three groups called A, B, C. Among the 7 examined genes, only those belonging to the HOX9-14 and the CDX groups exhibit a restricted expression along the oral-aboral axis during development and in the planula larva, while the others are expressed in very specialised areas at the medusa stage.

Conclusions/Significance

Cross species comparison reveals a strong variability of gene expression along the oral-aboral axis and during the life cycle among cnidarian lineages. The most parsimonious interpretation is that the Hox code, collinearity and conservative role along the antero-posterior axis are bilaterian innovations.