Independent Innexin Radiation Shaped Signaling in Ctenophores.

Innexins facilitate cell-cell communication by forming gap junctions or nonjunctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here, we identify and phylogenetically characterize the complete set of innexins of four ctenophores: Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, and Beroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of the M. leidyi innexin cluster suggests that this cluster has been maintained due to constraints related to gene regulation. We describe the basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

On the origin and evolution of RNA editing in metazoans.

Extensive adenosine-to-inosine (A-to-I) editing of nuclear-transcribed mRNAs is the hallmark of metazoan transcriptional regulation. Here, by profiling the RNA editomes of 22 species that cover major groups of Holozoa, we provide substantial evidence supporting A-to-I mRNA editing as a regulatory innovation originating in the last common ancestor of extant metazoans. This ancient biochemistry process is preserved in most extant metazoan phyla and primarily targets endogenous double-stranded RNA (dsRNA) formed by evolutionarily young repeats. We also find intermolecular pairing of sense-antisense transcripts as an important mechanism for forming dsRNA substrates for A-to-I editing in some but not all lineages. Likewise, recoding editing is rarely shared across lineages but preferentially targets genes involved in neural and cytoskeleton systems in bilaterians. We conclude that metazoan A-to-I editing might first emerge as a safeguard mechanism against repeat-derived dsRNA and was later co-opted into diverse biological processes due to its mutagenic nature.

Gene Loss may have Shaped the Cnidarian and Bilaterian Hox and ParaHox Complement.

Hox and ParaHox transcription factors are important for specifying cell fates along the primary body axes during the development of most animals. Within Cnidaria, much of the research on Hox/ParaHox genes has focused on Anthozoa (anemones and corals) and Hydrozoa (hydroids) and has concentrated on the evolution and function of cnidarian Hox genes in relation to their bilaterian counterparts. Here we analyze together the full complement of Hox and ParaHox genes from species representing all four medusozoan classes (Staurozoa, Cubozoa, Hydrozoa, and Scyphozoa) and both anthozoan classes (Octocorallia and Hexacorallia). Our results show that Hox genes involved in patterning the directive axes of anthozoan polyps are absent in the stem leading to Medusozoa. For the first time, we show spatial and temporal expression patterns of Hox and ParaHox genes in the upside-down jellyfish Cassiopea xamachana (Scyphozoa), which are consistent with diversification of medusozoan Hox genes both from anthozoans and within medusozoa. Despite unprecedented taxon sampling, our phylogenetic analyses, like previous studies, are characterized by a lack of clear homology between most cnidarian and bilaterian Hox and Hox-related genes. Unlike previous studies, we propose the hypothesis that the cnidarian-bilaterian ancestor possessed a remarkably large Hox complement and that extensive loss of Hox genes was experienced by both cnidarian and bilaterian lineages.

Cnidarian-Symbiodiniaceae symbiosis establishment is independent of photosynthesis.

Photosynthesis shapes the symbiotic relationships between cnidarians and Symbiodiniaceae algae-with many cnidarian hosts requiring symbiont photosynthate for survival-but little is known about how photosynthesis impacts symbiosis establishment. Here, we show that during symbiosis establishment, infection, proliferation, and maintenance can proceed without photosynthesis, but the ability to do so is dependent on specific cnidarian-Symbiodiniaceae relationships. The evaluation of 31 pairs of symbiotic relationships (five species of Symbiodiniaceae in sea anemone, coral, and jellyfish hosts) revealed that infection can occur without photosynthesis. A UV mutagenesis method for Symbiodiniaceae was established and used to generate six photosynthetic mutants that can infect these hosts. Without photosynthesis, Symbiodiniaceae cannot proliferate in the sea anemone Aiptasia or jellyfish Cassiopea but can proliferate in the juvenile polyps of the coral Acropora. After 6 months of darkness, Breviolum minutum is maintained within Aiptasia, indicating that Symbiodiniaceae maintenance can be independent of photosynthesis. Manipulating photosynthesis provides insights into cnidarian-Symbiodiniaceae symbiosis.

Adaptation to Bleaching: Are Thermotolerant Symbiodiniaceae Strains More Successful Than Other Strains Under Elevated Temperatures in a Model Symbiotic Cnidarian?

The ability of some symbiotic cnidarians to resist and better withstand stress factors that cause bleaching is a trait that is receiving increased attention. The adaptive bleaching hypothesis postulates that cnidarians that can form a stable symbiosis with thermotolerant Symbiodiniaceae strains may cope better with increasing seawater temperatures. We used polyps of the scyphozoan, Cassiopea xamachana, as a model system to test symbiosis success under heat stress. We sought to determine: (1) if aposymbiotic C. xamachana polyps could establish and maintain a symbiosis with both native and non-native strains of Symbiodiniaceae that all exhibit different tolerances to heat, (2) whether polyps with these newly acquired Symbiodiniaceae strains would strobilate (produce ephyra), and (3) if thermally tolerant Symbiodiniaceae strains that established and maintained a symbiosis exhibited greater success in response to heat stress (even if they are not naturally occurring in Cassiopea). Following recolonization of aposymbiotic C. xamachana polyps with different strains, we found that: (1) strains Smic, Stri, Slin, and Spil all established a stable symbiosis that promoted strobilation and (2) strains Bmin1 and Bmin2 did not establish a stable symbiosis and strobilation did not occur. Strains Smic, Stri, Slin, and Spil were used in a subsequent bleaching experiment; each of the strains was introduced to a subset of aposymbiotic polyps and once polyp tissues were saturated with symbionts they were subjected to elevated temperatures - 32°C and 34°C - for 2 weeks. Our findings indicate that, in general, pairings of polyps with Symbiodiniaceae strains that are native to Cassiopea (Stri and Smic) performed better than a non-native strain (Slin) even though this strain has a high thermotolerance. This suggests a degree of partner specificity that may limit the adaptive potential of certain cnidarians to increased ocean warming. We also observed that the free-living, non-native thermotolerant strain Spil was relatively successful in resisting bleaching during experimental trials. This suggests that free-living Symbiodiniaceae may provide a supply of potentially "new" thermotolerant strains to cnidarians following a bleaching event.

Perspectives on the expansion of human precision oncology and genomic approaches to sea turtle fibropapillomatosis.

Our recent Communications Biology research article revealed the genomic drivers and therapeutic vulnerabilities of sea turtle fibropapillomatosis tumors. Fibropapillomatosis is a debilitating tumorous disease afflicting populations of green sea turtles globally. While a virus is involved in the development of this disease, it is increasingly understood that the key trigger is linked to anthropogenic disturbances of the environment. The specific environmental co-trigger(s) has yet to be functionally confirmed. Here we outline the next steps required to advance our understanding of this enigmatic disease, to enable us to more effectively clinically combat it and to ultimately tackle its environmental co-trigger to halt and hopefully reverse the spread of fibropapillomatosis.

Sea turtle fibropapilloma tumors share genomic drivers and therapeutic vulnerabilities with human cancers.

Wildlife populations are under intense anthropogenic pressures, with the geographic range of many species shrinking, dramatic reductions in population numbers and undisturbed habitats, and biodiversity loss. It is postulated that we are in the midst of a sixth (Anthropocene) mass extinction event, the first to be induced by human activity. Further, threatening vulnerable species is the increased rate of emerging diseases, another consequence of anthropogenic activities. Innovative approaches are required to help maintain healthy populations until the chronic underlying causes of these issues can be addressed. Fibropapillomatosis in sea turtles is one such wildlife disease. Here, we applied precision-medicine-based approaches to profile fibropapillomatosis tumors to better understand their biology, identify novel therapeutics, and gain insights into viral and environmental triggers for fibropapillomatosis. We show that fibropapillomatosis tumors share genetic vulnerabilities with human cancer types, revealing that they are amenable to treatment with human anti-cancer therapeutics.

CRISPR knockouts reveal an endogenous role for ancient neuropeptides in regulating developmental timing in a sea anemone.

Neuropeptides are evolutionarily ancient peptide hormones of the nervous and neuroendocrine systems, and are thought to have regulated metamorphosis in early animal ancestors. In particular, the deeply conserved Wamide family of neuropeptides-shared across Bilateria (e.g. insects and worms) and its sister group Cnidaria (e.g. jellyfishes and corals)-has been implicated in mediating life-cycle transitions, yet their endogenous roles remain poorly understood. By using CRISPR-Cas9-mediated reverse genetics, we show that cnidarian Wamide-referred to as GLWamide-regulates the timing of life cycle transition in the sea anemone cnidarian Nematostella vectensis. We find that mutant planula larvae lacking GLWamides transform into morphologically normal polyps at a rate slower than that of the wildtype control larvae. Treatment of GLWamide null mutant larvae with synthetic GLWamide peptides is sufficient to restore a normal rate of metamorphosis. These results demonstrate that GLWamide plays a dispensable, modulatory role in accelerating metamorphosis in a sea anemone.

Germ layer-specific regulation of cell polarity and adhesion gives insight into the evolution of mesoderm.

In triploblastic animals, Par-proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par-proteins are degraded in all cells in the bifunctional gastrodermal epithelium. Using immunohistochemistry, CRISPR/Cas9 mutagenesis, and mRNA overexpression, we describe the functional association between Par-proteins, ß-catenin, and snail transcription factor genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß-catenin in the ectoderm by stabilizing its role in cell-adhesion, and that endomesodermal epithelial cells are organized by a different cell-adhesion system than overlying ectoderm. We also show that ectopic expression of snail genes, which are expressed in mesodermal derivatives in bilaterians, is sufficient to downregulate Par-proteins and translocate ß-catenin from the junctions to the cytoplasm in ectodermal cells. These data provide molecular insight into the evolution of epithelial structure and distinct cell behaviors in metazoan embryos.

Precision wildlife medicine: applications of the human-centred precision medicine revolution to species conservation.

The current species extinction crisis is being exacerbated by an increased rate of emergence of epizootic disease. Human-induced factors including habitat degradation, loss of biodiversity and wildlife population reductions resulting in reduced genetic variation are accelerating disease emergence. Novel, efficient and effective approaches are required to combat these epizootic events. Here, we present the case for the application of human precision medicine approaches to wildlife medicine in order to enhance species conservation efforts. We consider how the precision medicine revolution, coupled with the advances made in genomics, may provide a powerful and feasible approach to identifying and treating wildlife diseases in a targeted, effective and streamlined manner. A number of case studies of threatened species are presented which demonstrate the applicability of precision medicine to wildlife conservation, including sea turtles, amphibians and Tasmanian devils. These examples show how species conservation could be improved by using precision medicine techniques to determine novel treatments and management strategies for the specific medical conditions hampering efforts to restore population levels. Additionally, a precision medicine approach to wildlife health has in turn the potential to provide deeper insights into human health and the possibility of stemming and alleviating the impacts of zoonotic diseases. The integration of the currently emerging Precision Medicine Initiative with the concepts of EcoHealth (aiming for sustainable health of people, animals and ecosystems through transdisciplinary action research) and One Health (recognizing the intimate connection of humans, animal and ecosystem health and addressing a wide range of risks at the animal-human-ecosystem interface through a coordinated, collaborative, interdisciplinary approach) has great potential to deliver a deeper and broader interdisciplinary-based understanding of both wildlife and human diseases.

Ether-à-go-go family voltage-gated K+ channels evolved in an ancestral metazoan and functionally diversified in a cnidarian-bilaterian ancestor.

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K(+) channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg(2+) in Eag channels and Ca(2+) in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype.

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr.

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K(+) channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K(+) channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.

Aryl hydrocarbon receptor (AHR) in the cnidarian Nematostella vectensis: comparative expression, protein interactions, and ligand binding.

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachealess). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, ß-naphthoflavone). Together, our data suggest that AHR's original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian-bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates.

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis.

The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral-aboral (O-Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O-Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3ß inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O-Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal-vegetal patterning at earlier stages of anthozoan development.

Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis.

BACKGROUND: The contribution of cell proliferation to regeneration varies greatly between different metazoan models. Planarians rely on pluripotent neoblasts and amphibian limb regeneration depends upon formation of a proliferative blastema, while regeneration in Hydra can occur in the absence of cell proliferation. Recently, the cnidarian Nematostella vectensis has shown potential as a model for studies of regeneration because of the ability to conduct comparative studies of patterning during embryonic development, asexual reproduction, and regeneration. The present study investigates the pattern of cell proliferation during the regeneration of oral structures and the role of cell proliferation in this process. RESULTS: In intact polyps, cell proliferation is observed in both ectodermal and endodermal tissues throughout the entire oral-aboral axis, including in the tentacles and physa. Following bisection, there is initially little change in proliferation at the wound site of the aboral fragment, however, beginning 18 to 24 hours after amputation there is a dramatic increase in cell proliferation at the wound site in the aboral fragment. This elevated level of proliferation is maintained throughout the course or regeneration of oral structures, including the tentacles, the mouth, and the pharynx. Treatments with the cell proliferation inhibitors hydroxyurea and nocodazole demonstrate that cell proliferation is indispensable for the regeneration of oral structures. Although inhibition of regeneration by nocodazole was generally irreversible, secondary amputation reinitiates cell proliferation and regeneration. CONCLUSIONS: The study has found that high levels of cell proliferation characterize the regeneration of oral structures in Nematostella, and that this cell proliferation is necessary for the proper progression of regeneration. Thus, while cell proliferation contributes to regeneration of oral structures in both Nematostella and Hydra, Nematostella lacks the ability to undergo the compensatory morphallactic mode of regeneration that characterizes Hydra. Our results are consistent with amputation activating a quiescent population of mitotically competent stem cells in spatial proximity to the wound site, which form the regenerated structures.

The evolutionary diversification of LSF and Grainyhead transcription factors preceded the radiation of basal animal lineages.

BACKGROUND: The transcription factors of the LSF/Grainyhead (GRH) family are characterized by the possession of a distinctive DNA-binding domain that bears no clear relationship to other known DNA-binding domains, with the possible exception of the p53 core domain. In triploblastic animals, the LSF and GRH subfamilies have diverged extensively with respect to their biological roles, general expression patterns, and mechanism of DNA binding. For example, Grainyhead (GRH) homologs are expressed primarily in the epidermis, and they appear to play an ancient role in maintaining the epidermal barrier. By contrast, LSF homologs are more widely expressed, and they regulate general cellular functions such as cell cycle progression and survival in addition to cell-lineage specific gene expression. RESULTS: To illuminate the early evolution of this family and reconstruct the functional divergence of LSF and GRH, we compared homologs from 18 phylogenetically diverse taxa, including four basal animals (Nematostella vectensis, Vallicula multiformis, Trichoplax adhaerens, and Amphimedon queenslandica), a choanoflagellate (Monosiga brevicollis) and several fungi. Phylogenetic and bioinformatic analyses of these sequences indicate that (1) the LSF/GRH gene family originated prior to the animal-fungal divergence, and (2) the functional diversification of the LSF and GRH subfamilies occurred prior to the divergence between sponges and eumetazoans. Aspects of the domain architecture of LSF/GRH proteins are well conserved between fungi, choanoflagellates, and metazoans, though within the Metazoa, the LSF and GRH families are clearly distinct. We failed to identify a convincing LSF/GRH homolog in the sequenced genomes of the algae Volvox carteri and Chlamydomonas reinhardtii or the amoebozoan Dictyostelium purpureum. Interestingly, the ancestral GRH locus has become split into two separate loci in the sea anemone Nematostella, with one locus encoding a DNA binding domain and the other locus encoding the dimerization domain. CONCLUSIONS: In metazoans, LSF and GRH proteins play a number of roles that are essential to achieving and maintaining multicellularity. It is now clear that this protein family already existed in the unicellular ancestor of animals, choanoflagellates, and fungi. However, the diversification of distinct LSF and GRH subfamilies appears to be a metazoan invention. Given the conserved role of GRH in maintaining epithelial integrity in vertebrates, insects, and nematodes, it is noteworthy that the evolutionary origin of Grh appears roughly coincident with the evolutionary origin of the epithelium.

The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution.

Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ("hedge") domain and an autocatalytic intein ("hog") domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. We have identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, we have identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFbeta, FGF, and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp. Cnidarians represent a diverse group of animals with a predominantly epithelial body plan, and perhaps selective pressures to pattern epithelia resulted in the ontogeny of the hedgehog pathway in the common ancestor of the Cnidaria and Bilateria.

The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution.

Polyplacophorans, or chitons, are an important group of molluscs, which are argued to have retained many plesiomorphic features of the molluscan body plan. Polyplacophoran trochophore larvae posses several features that are distinctly different from those of their sister trochozoan taxa, including modifications of the ciliated prototrochal cells, the postrochal position of the larval eyes or ocelli, epidermal calcareous spicules, and a collection of serially reiterated epidermal shell plates. Despite these differences, chitons demonstrate a canonical pattern of equal spiral cleavage shared by other spiralian phyla that permits the identification of homologous cells across this animal clade. Cell lineage analysis using intracellular labeling on one chiton species, Chaetopleura apiculata, shows that the ocelli are generated from different lineal precursors (second-quartet micromeres: 2a, 2c) compared to those in all other spiralians studied to date (first-quartet micromeres: 1a, 1c). This situation implies that significant changes have also occurred in terms of the inductive interactions that control eye development in the spiralians. Although radical departures from the spiralian developmental program are seen in some molluscs (i.e., cephalopods), the findings presented here indicate that important changes can occur even within the highly constrained framework of the spiral cleavage program. Among spiralians, variation has been reported for the origin of the anterior, sensory, apical organ, which arises from the 1c and 1d micromeres in C. apiculata. The prototroch of C. apiculata consists of two to three irregular rows of ciliated cells but arise from 1q and 2q daughters, similar to that of Ischnochiton rissoi, as well as the gastropod, Patella vulgata. Despite certain early claims, there is no supporting evidence that any of the shell plates arise pretrochally in C. apiculata. The first seven of eight definitive shell plates that arise in the larva originate from shell secreting grooves in the postrochal region (derived from 2c, 2d, 3d). Earlier descriptions indicate that the eighth plate arises later at metamorphosis, and as this is formed posteriorly, it too forms in the postrochal region. On the other hand, epidermal spicules originate from both pretrochal and postrochal cells (1a,1d, 2a, 2c, 3c, 3d). The significance of these observations is discussed in light of various hypotheses concerning the origin of the conchiferan shell. This study reveals conservation, as well as evolutionary novelty, in the assignment of specific cell fates in the spiralians.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

Early evolution of a homeobox gene: the parahox gene Gsx in the Cnidaria and the Bilateria.

Homeobox transcription factors are commonly involved in developmental regulation in diverse eukaryotes, including plants, animals, and fungi. The origin of novel homeobox genes is thought to have contributed to many evolutionary innovations in animals. We perform a molecular phylogenetic analysis of cnox2, the best studied homeobox gene from the phylum Cnidaria, a very ancient lineage of animals. Among three competing hypotheses, our analysis decisively favors the hypothesis that cnox2 is orthologous to the gsx gene of Bilateria, thereby establishing the existence of this specific homeobox gene in the eumetazoan stem lineage, some 650-900 million years ago. We assayed the expression of gsx in the planula larva and polyp of the sea anemone Nematostella vectensis using in situ hybridization and reverse transcriptase polymerase chain reaction. The gsx ortholog of Nematostella, known as anthox2, is expressed at high levels in the posterior planula and the corresponding "head" region of the polyp. It cannot be detected in the anterior planula or the corresponding "foot" region of the polyp. We have attempted to reconstruct the evolution of gsx spatiotemporal expression in cnidarians and bilaterians using a phylogenetic framework. Because of the surprisingly high degree of variability in gsx expression within the Cnidaria, it is currently not possible to infer unambiguously the ancestral cnidarian condition or the ancestral eumetazoan condition for gsx expression.