BackgroundHox genes were critical to many morphological innovations of bilaterian animals. However, early Hox evolution remains obscure. Phylogenetic, developmental, and genomic analyses on the cnidarian sea anemone Nematostella vectensis challenge recent claims that the Hox code is a bilaterian invention and that no “true” Hox genes exist in the phylum Cnidaria.Methodology/Principal FindingsPhylogenetic analyses of 18 Hox-related genes from Nematostella identify putative Hox1, Hox2, and Hox9+ genes. Statistical comparisons among competing hypotheses bolster these findings, including an explicit consideration of the gene losses implied by alternate topologies. In situ hybridization studies of 20 Hox-related genes reveal that multiple Hox genes are expressed in distinct regions along the primary body axis, supporting the existence of a pre-bilaterian Hox code. Additionally, several Hox genes are expressed in nested domains along the secondary body axis, suggesting a role in “dorsoventral” patterning.Conclusions/SignificanceA cluster of anterior and posterior Hox genes, as well as ParaHox cluster of genes evolved prior to the cnidarian-bilaterian split. There is evidence to suggest that these clusters were formed from a series of tandem gene duplication events and played a role in patterning both the primary and secondary body axes in a bilaterally symmetrical common ancestor. Cnidarians and bilaterians shared a common ancestor some 570 to 700 million years ago, and as such, are derived from a common body plan. Our work reveals several conserved genetic components that are found in both of these diverse lineages. This finding is consistent with the hypothesis that a set of developmental rules established in the common ancestor of cnidarians and bilaterians is still at work today.

In recent years, a handful of model systems from the basal metazoan phylum Cnidaria have emerged to challenge long-held views on the evolution of animal complexity. The most-recent, and in many ways most-promising addition to this group is the starlet sea anemone, Nematostella vectensis. The remarkable amenability of this species to laboratory manipulation has already made it a productive system for exploring cnidarian development, and a proliferation of molecular and genomic tools, including the currently ongoing Nematostella genome project, further enhances the promise of this species. In addition, the facility with which Nematostella populations can be investigated within their natural ecological context suggests that this model may be profitably expanded to address important questions in molecular and evolutionary ecology. In this review, we explore the traits that make Nematostella exceptionally attractive as a model organism, summarize recent research demonstrating the utility of Nematostella in several different contexts, and highlight a number of developments likely to further increase that utility in the near future.

Nuclear sequence data, often from multiple loci, are increasingly being employed in analyses of population structure and history, yet there has been relatively little evaluation of methods for accurately and efficiently separating the alleles or haplotypes in heterozygous individuals. We compared the performance of a computational method of haplotype reconstruction and standard cloning methods using a highly variable intron (ornithine decarboxylase, intron 6) in three closely related species of dabbling ducks (genus Anas). Cloned sequences from 32 individuals were compared to results obtained from phase 2.1.1 . phase correctly identified haplotypes in 28 of 30 heterozygous individuals when the underlying model assumed no recombination. Haplotypes of the remaining two individuals were also inferred correctly except for unique polymorphisms, the phase of which was appropriately indicated as uncertain (phase probability = 0.5). For a larger set of 232 individuals, results were essentially identical regardless of the recombination model used and haplotypes for all 30 of the tested heterozygotes were correctly inferred, with the exception of uncertain phase for unique polymorphisms in one individual. In contrast, initial sequences of one clone per sample yielded accurate haplotype determination in only 26 of 30 individuals; polymerase chain reaction (PCR)/cloning errors resulting from misincorporation of individual nucleotides could be recognized and avoided by comparison to direct sequences, but errors due to PCR recombination resulted in incorrect haplotype reconstruction in four individuals. The accuracy of haplotypes reconstructed by phase, even when dealing with a relatively small number of samples and numerous variable sites, suggests broad utility of computational approaches for reducing the cost and improving the efficiency of data collection from nuclear sequence loci.

Homeoboxes of the cnidarian-bilaterian ancestor

The first near-complete set of homeodomains from a non-bilaterian animal is described.

Otx homeodomain transcription factors have been studied in a variety of eumetazoan animals where they have roles in anterior neural development, endomesoderm formation, and the formation of larval ciliated fields. Here, we describe the gene structure and developmental expression of three Otx loci in the starlet sea anemone, Nematostella vectensis (phylum Cnidaria; class Anthozoa). Nematostella's three Otx genes (OtxA, OtxB, and OtxC) are located in a compact genomic cluster spanning 63.6 kb. The homeodomains of all three Otx genes are highly similar to their bilaterian counterparts, but only OtxB exhibits the conserved WSP motif that is located downstream of the homeodomain in many Otx proteins. The genomic organization, in concert with phylogenetic analyses, indicates that two tandem duplications occurred in the lineage leading to Nematostella some time after the Cnidaria diverged from the Bilateria. In situ hybridization reveals that otx is initially expressed by invaginating mesendodermal cells in the gastrula. Later, each of the three otx paralogs is expressed in three discrete larval body regions: in the endoderm of the foot or physa, in an endodermal ring surrounding the pharynx, and in the ectoderm of the tentacles. These data suggest that a single otx locus had already acquired diverse developmental functions in the cnidarian-bilaterian ancestor. Furthermore, following two gene duplications in the line leading to Nematostella, there have been only minor alterations in the spatiotemporal expression of the three Otx paralogs. However, the absence of a conserved protein domain in OtxA and OtxC suggests functional evolution of the protein itself. J. Exp. Zool. (Mol. Dev. Evol.) 308B: 494-506, 2007. r 2007 How to cite this article: Mazza ME, Pang K, Martindale MQ, Finnerty JR. 2007. Genomic organization, gene structure, and developmental expression of three clustered otx genes in the sea anemone Nematostella vectensis. J. Exp. Zool. (Mol. Dev. Evol.) 308B: 494-506.

Background: Homeobox genes are a superclass of transcription factors with diverse developmental regulatory functions, which are found in plants, fungi and animals. In animals, several Antennapedia (ANTP)-class homeobox genes reside in extremely ancient gene clusters (for example, the Hox, ParaHox, and NKL clusters) and the evolution of these clusters has been implicated in the morphological diversification of animal bodyplans. By contrast, similarly ancient gene clusters have not been reported among the other classes of homeobox genes (that is, the LIM, POU, PRD and SIX classes).Results: Using a combination of in silico queries and phylogenetic analyses, we found that a cluster of three PRD-class homeobox genes (Homeobrain (hbn), Rax (rx) and Orthopedia (otp)) is present in cnidarians, insects and mollusks (a partial cluster comprising hbn and rx is present in the placozoan Trichoplax adhaerens). We failed to identify this 'HRO' cluster in deuterostomes; in fact, the Homeobrain gene appears to be missing from the chordate genomes we examined, although it is present in hemichordates and echinoderms. To illuminate the ancestral organization and function of this ancient cluster, we mapped the constituent genes against the assembled genome of a model cnidarian, the sea anemone Nematostella vectensis, and characterized their spatiotemporal expression using in situ hybridization. In N. vectensis, these genes reside in a span of 33 kb with the same gene order as previously reported in insects. Comparisons of genomic sequences and expressed sequence tags revealed the presence of alternative transcripts of Nv-otp and two highly unusual protein-coding polymorphisms in the terminal helix of the Nv-rx homeodomain. A population genetic survey revealed the Rx polymorphisms to be widespread in natural populations. During larval development, all three genes are expressed in the ectoderm, in non-overlapping territories along the oral-aboral axis, with distinct temporal expression. Conclusion:We report the first evidence for a PRD-class homeobox cluster that appears to have been conserved since the time of the cnidarian-bilaterian ancestor, and possibly even earlier, given the presence of a partial cluster in the placozoan Trichoplax. Very similar clusters comprising these three genes exist in Nematostella and diverse protostomes. Interestingly, in chordates, one member of the ancestral cluster (homeobrain) has apparently been lost, and there is no linkage between rx and orthopedia in any of the vertebrates. In Nematostella, the spatial expression of these three genes along the body column is not colinear with their physical order in the cluster but the temporal expression is, therefore, using the terminology that has been applied to the Hox cluster genes, the HRO cluster would appear to exhibit temporal but not spatial colinearity. It remains to be seen whether the mechanisms responsible for the evolutionary conservation of the HRO cluster are the same mechanisms responsible for cohesion of the Hox cluster and other ANT...

Background: Msx originated early in animal evolution and is implicated in human genetic disorders. To reconstruct the functional evolution of Msx and inform the study of human mutations, we analyzed the phylogeny and synteny of 46 metazoan Msx proteins and tracked the duplication, diversification and loss of conserved motifs.