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.     

The origins of axial patterning in the metazoa: how old is bilateral symmetry?

Bilateral symmetry is a hallmark of the Bilateria. It is achieved by the intersection of two orthogonal axes of polarity: the anterior-posterior (A-P) axis and the dorsal-ventral (D-V) axis. It is widely thought that bilateral symmetry evolved in the common ancestor of the Bilateria. However, it has long been known that members of the phylum Cnidaria, an outgroup to the Bilateria, also exhibit bilateral symmetry. Recent studies have examined the developmental expression of axial patterning genes in members of the phylum Cnidaria. Hox genes play a conserved role in patterning the A-P axis of bilaterians. Hox genes are expressed in staggered axial domains along the oral-aboral axis of cnidarians, suggesting that Hox patterning of the primary body axis was already present in the cnidarian-bilaterian ancestor. Dpp plays a conserved role patterning the D-V axis of bilaterians. Asymmetric expression of dpp about the directive axis of cnidarians implies that this patterning system is similarly ancient. Taken together, these result imply that bilateral symmetry had already evolved before the Cnidaria diverged from the Bilateria.     

Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms

As a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems.     

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.     

Expansion of the SOX gene family predated the emergence of the Bilateria

Members of the SOX gene family are involved in regulating many developmental processes including neuronal determination and differentiation, and in carcinogenesis. So far they have only been identified in species from the Bilateria (deuterostomes and protostomes). To understand the origins of the SOX family, we used a PCR-based strategy to obtain 28 new sequences of SOX gene HMG domains from four non-bilaterian Metazoa: two sponge species, one ctenophore and one cnidarian. One additional SOX sequence was retrieved from EST sequences of the cnidarian species Clytia hemisphaerica. Unexpected SOX gene diversity was found in these species, especially in the cnidarian and the ctenophore. The topology of gene relationships deduced by Maximum Likelihood analysis, although not supported by bootstrap values, suggested that the SOX family started to diversify in the metazoan stem branch prior to the divergence of demosponges, and that further diversification occurred in the eumetazoan branch, as well as later in calcisponges, ctenophores, cnidarians and vertebrates. In contrast, gene loss appears to have occurred in the nematode and probably in other protostome lineages, explaining their lower number of SOX genes.     

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.     

Posterior expression of nanos orthologs during embryonic and larval development of the anthozoan Nematostella vectensis

Cnidarians are primitive animals located in a basal position in the phylogenetic tree of the Animal Kingdom, as an outgroup of the Bilaterians. Therefore, studies on cnidarian developmental biology may illustrate how fundamental developmental processes have originated and changed during animal evolution. A particular example of this is the establishment of polarity along the body axes, which is under the control of a number of developmental genes, most of them conserved in evolution and playing similar roles in diverged species. Concerning the anterior-posterior axis, genetic and molecular studies on Drosophila have shown that the nanos gene plays an essential role in defining posterior structures during early embryonic development. Here we report the isolation of two nanos orthologs in the anthozoan Nematostella vectensis. We show that nanos mRNA is asymmetrically distributed in the fertilized egg and this asymmetry is maintained during embryonic development. At gastrula and planula larva stages, nanos expression is permanently associated with posterior body regions. These results, together with our previous analysis in the hydrozoan Podocoryne carnea, indicate that posterior nanos expression during development is a conserved feature among cnidarians. Therefore, the potential role of cnidarian nanos in defining axial polarity as a posterior determinant would represent an ancestral trait in the Animal Kingdom.     

Colonial origin for Emetazoa: major morphological transitions and the origin of bilaterian complexity

A new hypothesis for the evolution of Bilateria is presented. It is based on a reinterpretation of the morphological characters shared by protostomes and deuterostomes, which, when taken together with developmental processes shared by the two lineages, lead to the inescapable conclusion that the last common ancestor of Bilateria was complex. It possessed a head, a segmented trunk, and a tail. The segmented trunk was further divided into two sections. A dorsal brain innervated one or more sensory cells, which included photoreceptors. "Appendages" or outgrowths were present. The bilaterian ancestor also possessed serially repeated "segments" that were expressed ontogenetically as blocks of mesoderm or somites with adjoining fields of ectoderm or neuroectoderm. It displayed serially repeated gonads (gonocoels), each with a gonoduct and gonopore to the exterior, and serially repeated "coeloms" with connections to both the gut and the exterior (gill slits and pores). Podocytes, some of which were serially repeated in the trunk, formed sites of ultrafiltration. In addition, the bilaterian ancestor had unsegmented coeloms and a contractile blood vessel or "heart" formed by coelomic myoepithelial cells. These cells and their underlying basement membrane confine the hemocoelic fluid, or blood, in the connective tissue compartment. A possible scenario to account for this particular suite of characters is one in which a colony of organisms with a cnidarian grade of organization became individuated into a new entity with a bilaterian grade of organization. The transformation postulated encompassed three major transitions in the evolution of animals. These transitions included the origins of Metazoa, Eumetazoa, and Bilateria and involved the successive development of poriferan, cnidarian, and bilaterian grades of organization. Two models are presented for the sponge-to-cnidarian transition. In both models the loss of a flow-through pattern of water circulation in poriferans and the establishment of a single opening and epithelia sensu stricto in cnidarians are considered crucial events. In the model offered for the cnidarian-to-bilaterian transition, the last common ancestor of Eumetazoa is considered to have had a colonial, cnidarian-grade of organization. The ancestral cnidarian body plan would have been similar to that exhibited by pennatulacean anthozoans. It is postulated that a colonial organization could have provided a preadaptive framework for the evolution of the complex and modularized body plan of the triploblastic ancestor of Bilateria. Thus, one can explore the possibility that problematica such as ctenophores, the Ediacaran biota, archaeocyaths, and Yunnanozoon reflect the fact that complexity originated early and involved the evolution of a macroscopic compartmented ancestor. Bilaterian complexity can be understood in terms of Beklemishev "cycles" of duplication and colony individuation. Two such cycles appear to have transpired in the early evolution of Metazoa. The first gave rise to a multicellular organism with a sponge grade of organization and the second to the modularized ancestor of Bilateria. The latter episode may have been favored by the ecological conditions in the late Proterozoic. Whatever its cause, the individuation of a cnidarian-grade colony furnishes a possible explanation for the rapid diversification of bilaterians in the late Vendian and Cambrian. The creation of a complex yet versatile prototype, which could be rapidly modified by selection into a profusion of body plans, is postulated to have affected the timing, mode, and extent of the "Cambrian explosion." During the radiations, selective loss or simplification may have been as creative a force as innovation. Finally, colony individuation may have been a unique historical event that imprinted the development of bilaterians as the zootype and phylotypic stage. (ABSTRACT TRUNCATED)     

Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellyfish: a connection to the mesoderm of bilateria

One major difference between simple metazoans such as cnidarians and all the bilaterian animals is thought to involve the invention of mesoderm. The terms diploblasts and triploblasts are therefore, often used to group prebilaterian and bilaterian animals, respectively. However, jellyfish contain well developed striated and smooth muscle tissues that derive from the entocodon, a mesoderm-like tissue formed during medusa development. We investigated the hypothesis, that the entocodon could be homologous to the third germ layer of bilaterians by analyzing the structures and expression patterns of the homologues of Brachyury, Mef2, and Snail in the jellyfish Podocoryne carnea. These are regulatory genes from the T-box, MADS-box and zinc finger families known to play important roles in bilaterian mesoderm patterning and muscle differentiation. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. Based on these data we conclude that the common ancestor of the cnidarians and bilaterians not only shared genes that play a role in regulating myogenesis but already used them to develop and differentiate muscle systems similar to those of triploblasts.     

Back in time: a new systematic proposal for the Bilateria

Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.     

Tandem organization of independently duplicated homeobox genes in the basal cnidarian <Emphasis Type="Italic">Acropora millepora</Emphasis>

A number of examples of independently duplicated regulatory genes have been identified in cnidarians, but the extent of this phenomenon and organization of these duplicated genes are unknown. Here we describe the identification of three pairs of independently duplicated homeobox genes in the anthozoan cnidarian, Acropora millepora. In each case, the pairs of paralogous genes are tightly linked, but the extent of sequence divergence implies that these do not reflect recent duplication events. The phenomenon is likely to be more general, as the examples reported here represent most of the limited number of Acropora homeobox genes for which genomic data are yet available.N. Hislop and D. de Jong contributed equally to this workEdited by M. Akam     

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.