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.     

Colony integration and the expression of the Hox gene, Cnox-2, in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa).

The stolonal mat is an anatomical feature correlated with increased colonial integration in several lineages of the cnidarian class Hydrozoa. Cnox-2 is a Hox gene known to be expressed in the body column of the cnidarian polyp. We report the pattern of Cnox-2 expression in both the stolonal mat and free stolons of the hydroid Hydractinia symbiolongicarpus. The gene is found to have high levels of expression in the mat similar to that found in the basal portion of the polyp, but it is not detectably expressed in those regions of free stolons where polyps are budded. These findings suggest that the stolonal mat arose via an expansion of the basal ectoderm of the polyp.     

The early ANTP gene repertoire: insights from the placozoan genome

The evolution of ANTP genes in the Metazoa has been the subject of conflicting hypotheses derived from full or partial gene sequences and genomic organization in higher animals. Whole genome sequences have recently filled in some crucial gaps for the basal metazoan phyla Cnidaria and Porifera. Here we analyze the complete genome of Trichoplax adhaerens, representing the basal metazoan phylum Placozoa, for its set of ANTP class genes. The Trichoplax genome encodes representatives of Hox/ParaHox-like, NKL, and extended Hox genes. This repertoire possibly mirrors the condition of a hypothetical cnidarian-bilaterian ancestor. The evolution of the cnidarian and bilaterian ANTP gene repertoires can be deduced by a limited number of cis-duplications of NKL and "extended Hox" genes and the presence of a single ancestral "ProtoHox" gene.     

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.     

The Hox gene complement of a pelagic chaetognath, Flaccisagitta enflata

Chaetognaths are transparent marine animals that are ubiquitous and abundant members of oceanic zooplanktonic communities. Their phylogenetic position within the Metazoa, however, has remained obscure since their discovery. Morphology and embryology have traditionally allied chaetognaths with deuterostomes, but molecular evidence suggests otherwise. Two recent multigene expressed sequence tag (EST) molecular phylogenomic studies suggest that chaetognaths are either sister to the Lophotrochozoa (Matus et al. 2006) or to all protostomes (Marlétaz et al. 2006). We have isolated eight Hox genes, one Parahox gene, and Mox, a related homeodomain gene, from the pelagic chaetognath, Flaccisagitta enflata. Although chaetognath central class Hox genes lack the Lox5 or "spiralian" parapeptide, a diagnostic amino-acid motif that has been utilized previously to assign lophotrochozoan affinity, they do possess a central class Hox gene that has a partial "Ubd-A peptide" found in both ecdysozoan and lophotrochozoan Ubx/Abd-A/Lox2/Lox4 genes. Additionally, we report the presence of two distinct chaetognath posterior Hox genes that possess both ecdysozoan and lophotrochozoan signature amino-acid motifs. The phylogenetic position of chaetognaths, as well as the evolution of the Hox cluster, is discussed in light of these data.     

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.     

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.     

Axial patterning and diversification in the cnidaria predate the Hox system

Across the animal kingdom, Hox genes are organized in clusters whose genomic organization reflects their central roles in patterning along the anterior/posterior (A/P) axis . While a cluster of Hox genes was present in the bilaterian common ancestor, the origins of this system remain unclear (cf. ). With new data for two representatives of the closest extant phylum to the Bilateria, the sea anemone Nematostella and the hydromedusa Eleutheria, we argue here that the Cnidaria predate the evolution of the Hox system. Although Hox-like genes are present in a range of cnidarians, many of these are paralogs and in neither Nematostella nor Eleutheria is an equivalent of the Hox cluster present. With the exception of independently duplicated genes, the cnidarian genes are unlinked and in several cases are flanked by non-Hox genes. Furthermore, the cnidarian genes are expressed in patterns that are inconsistent with the Hox paradigm. We conclude that the Cnidaria/Bilateria split occurred before a definitive Hox system developed. The spectacular variety in morphological and developmental characteristics shown by extant cnidarians demonstrates that there is no obligate link between the Hox system and morphological diversity in the animal kingdom and that a canonical Hox system is not mandatory for axial patterning.     

Consequences of Hox gene duplication in the vertebrates: an investigation of the zebrafish Hox paralogue group 1 genes

As a result of a whole genome duplication event in the lineage leading to teleosts, the zebrafish has seven clusters of Hox patterning genes, rather than four, as described for tetrapod vertebrates. To investigate the consequences of this genome duplication, we have carried out a detailed comparison of genes from a single Hox paralogue group, paralogue group (PG) 1. We have analyzed the sequences, expression patterns and potential functions of all four of the zebrafish PG1 Hox genes, and compared our data with that available for the three mouse genes. As the basic functions of Hox genes appear to be tightly constrained, comparison with mouse data has allowed us to identify specific changes in the developmental roles of Hox genes that have occurred during vertebrate evolution. We have found variation in expression patterns, amino acid sequences within functional domains, and potential gene functions both within the PG1 genes of zebrafish, and in comparison to mouse PG1 genes. We observed novel expression patterns in the midbrain, such that zebrafish hoxa1a and hoxc1a are expressed anterior to the domain traditionally thought to be under Hox patterning control. The hoxc1a gene shows significant coding sequence changes in known functional domains, which correlate with a reduced capacity to cause posteriorizing transformations. Moreover, the hoxb1 duplicate genes have differing functional capacities, suggesting divergence after duplication. We also find that an intriguing function 'shuffling' between paralogues has occurred, such that one of the zebrafish hoxb1 duplicates, hoxb1b, performs the role in hindbrain patterning played in mouse by the non-orthologous Hoxa1 gene.     

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.     

Conservation of Hox/ParaHox-related genes in the early development of a cnidarian.

To clarify the relationship between axial patterning in cnidarians and bilaterians, we have investigated the embryonic development of the hydrozoan Podocoryne carnea. The expression of Hox-like homeobox genes was analyzed by RT-PCR and in situ hybridization. Cnox1-Pc, an anterior Hox gene, is a maternal message. It is present throughout larval development, first weakly in all blastomeres and later restricted mostly to the anterior pole of the planula. Gsx, an anterior ParaHox gene, is first seen in the anterior endoderm but also extends into posterior regions. Cnox4-Pc, an orphan Hox-like gene, is expressed in the egg as a ring-shaped cloud around the germinal vesicle. After fertilization, the message remains in most animal blastomeres. When the embryo elongates in late blastula, staining is restricted to a few cells at the posterior pole where gastrulation will start. However, once gastrulation starts, the Cnox4-Pc signal disappears and is absent in later stages of larval development. Phylogenetic analysis shows that not all cnidarian Hox-like genes have recognizable orthologues in bilaterian groups. However, the expression analysis of Cnox1-Pc and Gsx correlates to some extent with the expression pattern of cognate genes of bilaterians, confirming the conservation of genes involved in organizing animal body plans and their putative common ancestral origin.     

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.     

Position-specific activity of the Hox1.1 promoter in transgenic mice

During development, positional values have to be assigned to groups of cells. The murine Hox genes are a class of genes that are predicted to be involved at some stage in this process. During embryogenesis they are expressed in distinct overlapping region- and stage-specific patterns and therefore must be regulated in response to positional information. In this study, we have analysed the activity of Hox1.1 promoter sequences in transgenic mice. The use of lacZ as a marker allows a detailed analysis of expression at the single cell level during early embryonic development. We show that 3.6 kbp of promoter and 1.7 kbp of 3' sequences provide sufficient regulatory information to express a transgene in a spatial and temporal manner indistinguishable from the endogenous Hox1.1 gene during the period of development when Hox1.1 expression is established. The activation occurs in a strict order in specific ectodermal and mesodermal domains. Within each of these domains the transgene is activated over a period of four hours apparently randomly in single cells. In a following second period, Hox1.1 and transgene expression patterns diverge. In this period, transgene expression persists in many mesodermally derived cells that do not express Hox1.1 indicating the absence of a negative regulatory element in the transgene. The anterior boundary of transgene expression is identical to that of Hox1.1. However, no posterior boundary of transgene expression is set, suggesting that a separate element absent from the transgene specifies this boundary.     

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.     

Novel interactions between vertebrate Hox genes

Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.     

PCR survey of Hox genes in the crinoid and ophiuroid: evidence for anterior conservation and posterior expansion in the echinoderm Hox gene cluster

To help elucidate the cluster organization of Hox genes in echinoderms, we amplified a homeobox region by polymerase chain reaction (PCR) and cloned and sequenced the PCR products for the comatulid crinoid Oxycomanthus japonicus and the ophiuroid Stegophiura sladeni. The crinoid had at least three anterior, four medial, and four posterior genes, and the ophiuroid had at least one anterior, three medial, and six (one of which being a possible trans-paralog) posterior genes. The survey of the crinoid detected all three anterior complements (PG1-3 genes). It was inferred that the Hox genes of each species are organized into a single cluster and that a novel cognate group of echinoderm posterior genes (tentatively termed HboxP9) exists among echinoderms in addition to the known posterior genes Hbox4, Hbox7, and Hbox10. The results, combined with the data of other echinoderm classes, strongly suggest that the presence of a single Hox gene cluster is a common feature among echinoderms and that the cluster has the general features of the deuterostome Hox gene cluster, i.e., the anterior conservation and posterior expansion. The results of the ophiuroid imply that the posterior genes in this class diversified after the phylum Echinodermata had been established.