Cnidarians both occupy a key evolutionary position and are ecologically important as predators, prey and structure-builders in marine and freshwater environments. As "basal" metazoans, cnidarians form an outgroup to the bilaterian animals and are intermediate in complexity between sponges and bilaterians. In spite of the acknowledged importance of understanding cnidarians from both an ecological and an evolutionary perspective, cnidarian physiology is poorly understood, particularly at the molecular level. Thus, characterizing cnidarian NRs provides insight both into evolution of NR signaling and bioregulatory processes in a major group of aquatic animals.
Homology and expression of NvNRs
Analysis of the phylogenetic relationships among NRs from the starlet sea anemone, Nematostella vectensis, provides strong support for the diversification of NR family 2 prior to the divergence of the cnidarian and bilaterian lineages. Phylogenetic analyses of N. vectensis and bilaterian NRs revealed cnidarian orthologs of HNF4, COUP, TLL, and TR2/4. Grasso et al.  identified 10 NRs in the coral Acropora millepora, including several members of NR family 2. Our results support their conclusion that family 2 was well diversified prior to the split between the cnidarian and bilaterian lineages.
NvNR4 is an apparent homolog of hepatocyte nuclear factor 4 (HNF4, subfamily 2A), which has also been identified in vertebrates, insects, nematodes, and corals [4, 22, 23]. In mammals, HNF4α binds endogenous fatty acid ligands [24, 25] and regulates hepatocyte differentiation, energetic metabolism and xenobiotic detoxification. HNF4 also regulates insect gut development [26, 27]. HNF4 underwent extensive duplications in C. elegans  such that inferring homologous functions for HNF4 from the nematode is difficult. One C. elegans paralog, nhr-49, is involved in energetic metabolism as a regulator of fat storage . In addition, an HNF4 homolog has been cloned from sponges and may be similar to the ancestral NR . Sponge HNF4 is expressed throughout development in ciliated column cells of the outer epithelium . In N. vectensis, NvNR4 was expressed at all developmental time points with higher mean expression from larval to adult stages. We predict that NvNR4 will be expressed in endodermal tissue, which would be consistent with conserved roles in development of the digestive epithelium, energetic metabolism, and/or detoxification.
NvNR5- NvNR9 cluster with subfamily 2E (TLL/TLX, FAX, PNR); members of this subfamily have previously been reported in corals [4, 30]. In our phylogenetic analyses, we were unable to fully resolve the evolutionary relationships of N. vectensis and bilaterian NRs within subfamily 2E (Figure 2). In other animals, members of subfamily 2E are involved in neural differentiation. Tailless homologs in mammals (TLX) and insects (TLL) are particularly important for eye and forebrain development [31–34] and embryonic anterior-posterior patterning . FAX-1 regulates neural patterning in C. elegans [36, 37], and PNR plays a more specialized role by regulating retinal development in vertebrates . Several N. vectensis members of subfamily 2E (i.e., NvNR5, 7, 8) were highly expressed during early developmental stages coinciding with neurogenesis and embryonic patterning . The stage of maximal expression varied among members of this subfamily, indicating potential diversification of function within the cnidarian lineage.
NvNR10-NvNR14 group with subfamily 2F (COUP-TFs); COUP-TFs have also been identified in corals, hydra, flatworms, sea urchin, and lancelets [3, 4, 40–42]. NvNR10 was most closely related to bilaterian COUP-TFs, while NvNR11- NvNR14 are supported as an independent radiation with this subfamily. COUP-TFs generally act as transcriptional repressors and regulate development of muscles, the heart and the nervous system, particularly differentiation of the hindbrain and photoreceptors [43–46]. In hydra, COUP-TF is expressed in nematoblasts (nematocyte precursors) and in a subset of neuronal cells, consistent with a conserved role of COUP-TF in regulating neural differentiation . COUP-TFs can also affect reproductive processes through cross-talk with estrogen receptors and ecdysteroid receptors in vertebrates and insects, respectively [47–50]. COUP-TF homologs from N. vectensis showed diverse expression patterns during development. For example, NvNR12 and13 were primarily expressed during embryonic and/or early larval stages with little expression in the juvenile and adult stages. The expression of these COUP-TF-like genes coincides with neurogenesis and embryo patterning . In contrast, we observed relatively high expression of NvNR10 in adults but lower expression during embryogenesis and larval development.
We identified two members of the subfamily 2C/D (TR2/4, NvNR15-16). TR2/4 homologs have been identified in a range of animals including vertebrates, sea urchins, ascidians, nematodes, insects, flatworms, and corals [51–54]. Protostomes and most deuterostomes have a single TR2/4 homolog. Interestingly, both N. vectensis and the coral Acropora millepora  contain two TR2/4 homologs, suggesting a cnidarian-specific duplication. In general, TR2/4 homologs act as transcriptional repressors through several mechanisms including competition with other nuclear receptors for binding sites and co-factors [55, 56]. TR2/4 homologs are broadly expressed in vertebrate tissues  and throughout development in mammals , ascidians , and flatworms . We observed similar ubiquitous expression in N. vectensis where both NvNR15 and 16 were expressed throughout all developmental stages.
NvNR17 forms a monophyletic grouping with NR family 6 (GCNFs, germ cell nuclear factors). Although bootstrap support for this assignment is relatively weak, likelihood analyses of the DBD plus LBD and each of these domains individually group NvNR17 as a homolog to the GCNFs. While neighbor joining analysis placed NvNR17 as an outgroup to NR families 5 and 6, bootstrap support for deep nodes of this tree were low relative to the maximum likelihood analyses, potentially obscuring evolutionary relationships. GCNF has been reported from a number of bilaterian phyla. This is the first report of a putative cnidarian homolog from this NR family. Expression of GCNF homologs varies greatly among taxa. In D. melanogaster, DmHR4 is expressed following pulses of 20-hydroxyecdysone during discrete stages of embryogenesis, the second larval instar and prepupal development [59, 60]. In C. elegans, CeNHR-91 is expressed in response to ATP-binding cassette protein E in embryos, larvae and several adult tissues . In vertebrates, GCNF is expressed primarily expressed in gonadal tissues and during embryogenesis [62, 63]. In vertebrates, GCNF is involved in neuronal differentiation, germ cell development, and axial patterning, and gametogenesis [63–65]. In our study, NvNR17 expression increased throughout the sampled developmental stages. Highest expression was in adult tissues, which may reflect a role in gametogenesis.
NvNR1-3 did not group well with any previously defined NR family. These genes are most closely related to NR Family 1 and 4. With the available data, we cannot conclude whether these represent the descendents of ancient NRs that later diversified into recognized NR families or if these genes represent an independent radiation somewhere within the cnidarian lineage. Further sampling within the Cnidaria will be necessary to help resolve the evolutionary relationship of these NRs.
We did not identify a member of NR family 3 in N. vectensis. NR family 3 is represented in protostomes and deuterostomes [5, 21, 66, 67], and has been more recently identified in the placozoan Trichoplax adhaerens [, Reitzel and Tarrant, unpublished data] but has not yet been reported from any cnidarian. Thus, NR 3 homologs have apparently been lost early in the cnidarian lineage. NR family 3 contains the vertebrate-type steroid (i.e., non-ecdysteroid) receptors. While NR3 family members were not identified in N. vectensis, estrogens and other steroids have been detected in cnidarian tissues [12, 15, 69], are apparently released during spawning events [12, 69], and can experimentally affect coral growth and reproduction . The mechanism for steroid action in cnidarians is currently unknown and may be mediated through nuclear receptors that are not orthologs of the vertebrate steroid receptors or through alternative mechanisms.