TEMPERATURE AND SALINITY AFFECT DEVELOPMENT OF THE PARASITIC SEA ANEMONE EDWARDSIELLA LINEATA POTENTIALLY LIMITING ITS IMPACT AS A BIOLOGICAL CONTROL ON THE CTENOPHORE MNEMIOPSIS LEIDYI.

The lined sea anemone, Edwardsiella lineata, parasitizes the ctenophore Mnemiopsis leidyi, which is one of the most destructive marine invasive species in the world. Mnemiopsis leidyi is known to tolerate a wide range of environmental conditions. However, the environmental tolerances of its most prominent parasite have never been characterized. Here we determined the effects of temperature (18, 22, 26, and 30 C) and salinity (6, 15, 24, and 33 ppt) on the survival and development of E. lineata from a vermiform parasite to a free-living polyp. At higher temperatures and lower salinities, E. lineata experienced significantly higher mortality, and it failed to develop into an adult polyp at the highest temperature (30 C) and lowest salinities we tested (6 ppt or 15 ppt). While such temperature and salinity restrictions would not currently prevent E. lineata from infecting M. leidyi in many of the European waters where it has become a destructive invasive species, these environmental limitations may be reducing overlap between host and parasite within the host's native range, a situation that could be exacerbated by climate change.

The impact of autotrophic versus heterotrophic nutritional pathways on colony health and wound recovery in corals.

For animals that harbor photosynthetic symbionts within their tissues, such as corals, the different relative contributions of autotrophy versus heterotrophy to organismal energetic requirements have direct impacts on fitness. This is especially true for facultatively symbiotic corals, where the balance between host-caught and symbiont-produced energy can be altered substantially to meet the variable demands of a shifting environment. In this study, we utilized a temperate coral-algal system (the northern star coral, Astrangia poculata, and its photosynthetic endosymbiont, Symbiodinium psygmophilum) to explore the impacts of nutritional sourcing on the host's health and ability to regenerate experimentally excised polyps. For fed and starved colonies, wound healing and total colony tissue cover were differentially impacted by heterotrophy versus autotrophy. There was an additive impact of positive nutritional and symbiotic states on a coral's ability to initiate healing, but a greater influence of symbiont state on the recovery of lost tissue at the lesion site and complete polyp regeneration. On the other hand, regardless of symbiont state, fed corals maintained a higher overall colony tissue cover, which also enabled more active host behavior (polyp extension) and endosymbiont behavior (photosynthetic ability of Symbiondinium). Overall, we determined that the impact of nutritional state and symbiotic state varied between biological functions, suggesting a diversity in energetic sourcing for each of these processes.

Sex-specific and developmental expression of Dmrt genes in the starlet sea anemone, Nematostella vectensis.

BACKGROUND: The molecular mechanisms underlying sex determination and differentiation in animals are incredibly diverse. The Dmrt (doublesex and mab-3 related transcription factor) gene family is an evolutionary ancient group of transcription factors dating to the ancestor of metazoans that are, in part, involved in sex determination and differentiation in numerous bilaterian animals and thus represents a potentially conserved mechanism for differentiating males and females dating to the protostome-deuterostome ancestor. Recently, the diversity of this gene family throughout animals has been described, but the expression and potential function for Dmrt genes is not well understood outside the bilaterians. RESULTS: Here, we report sex- and developmental-specific expression of all 11 Dmrts in the starlet sea anemone Nematostella vectensis. Nine out of the eleven Dmrts showed significant differences in developmental expression, with the highest expression typically in the adult stage and, in some cases, with little or no expression measured during embryogenesis. When expression was compared in females and males, seven of the eleven Dmrt genes had significant differences in expression with higher expression in males than in females for six of the genes. Lastly, expressions of two Dmrt genes with differential expression in each sex are located in the mesenteries and into the pharynx in polyps. CONCLUSIONS: Our results show that the phylogenetic diversity of Dmrt genes in N. vectensis is matched by an equally diverse pattern of expression during development and in each sex. This dynamic expression suggests multiple functions for Dmrt genes likely present in early diverging metazoans. Detailed functional analyses of individual genes will inform hypotheses regarding the antiquity of function for these transcription factors.

Production of a reference transcriptome and transcriptomic database (EdwardsiellaBase) for the lined sea anemone, Edwardsiella lineata, a parasitic cnidarian.

BACKGROUND: The lined sea anemone Edwardsiella lineata is an informative model system for evolutionary-developmental studies of parasitism. In this species, it is possible to compare alternate developmental pathways leading from a larva to either a free-living polyp or a vermiform parasite that inhabits the mesoglea of a ctenophore host. Additionally, E. lineata is confamilial with the model cnidarian Nematostella vectensis, providing an opportunity for comparative genomic, molecular and organismal studies. DESCRIPTION: We generated a reference transcriptome for E. lineata via high-throughput sequencing of RNA isolated from five developmental stages (parasite; parasite-to-larva transition; larva; larva-to-adult transition; adult). The transcriptome comprises 90,440 contigs assembled from >15 billion nucleotides of DNA sequence. Using a molecular clock approach, we estimated the divergence between E. lineata and N. vectensis at 215-364 million years ago. Based on gene ontology and metabolic pathway analyses and gene family surveys (bHLH-PAS, deiodinases, Fox genes, LIM homeodomains, minicollagens, nuclear receptors, Sox genes, and Wnts), the transcriptome of E. lineata is comparable in depth and completeness to N. vectensis. Analyses of protein motifs and revealed extensive conservation between the proteins of these two edwardsiid anemones, although we show the NF-κB protein of E. lineata reflects the ancestral structure, while the NF-κB protein of N. vectensis has undergone a split that separates the DNA-binding domain from the inhibitory domain. All contigs have been deposited in a public database (EdwardsiellaBase), where they may be searched according to contig ID, gene ontology, protein family motif (Pfam), enzyme commission number, and BLAST. The alignment of the raw reads to the contigs can also be visualized via JBrowse. CONCLUSIONS: The transcriptomic data and database described here provide a platform for studying the evolutionary developmental genomics of a derived parasitic life cycle. In addition, these data from E. lineata will aid in the interpretation of evolutionary novelties in gene sequence or structure that have been reported for the model cnidarian N. vectensis (e.g., the split NF-κB locus). Finally, we include custom computational tools to facilitate the annotation of a transcriptome based on high-throughput sequencing data obtained from a "non-model system."

Evolutionary expression of glucose-dependent-insulinotropic polypeptide (GIP).

Glucose-dependent insulinotropic polypeptide (GIP) is a mammalian incretin hormone released into the circulation following nutrient ingestion. We examined the functional evolution of GIP and its relationship with insulin to delineate their respective roles in promoting nutrient efficiency. Expression patterns were examined in the sea lamprey (Petromyzon marinus), a basal vertebrate lacking a distinct pancreas, and in the zebrafish, Xenopus laevis, chicken, and mouse, organisms possessing extraintestinal pancreata. Although sea lamprey genomic analysis predicted a potential GIP-like gene, transcripts were not detected, and insulin expression was confined to the caudal pancreatic bud. GIP was detected in both the intestine and pancreas of the zebrafish and X. laevis. In contrast, GIP and insulin expression were limited to the intestine and pancreas, respectively, in chicken and mouse. Phylogenetic analysis of the glucagon-like ligands suggested proglucagon as the common ancestor, supporting the theory that GIP arose as a gene duplication of proglucagon. Insulin-secreting cells in the sea lamprey intestine may have obviated the need for an enteroinsular axis, and zebrafish may represent an evolutionary transition where GIP does not yet function as an incretin hormone. These observations are consistent with the hypothesis that GIP and insulin influence survival advantage by enhancing the efficiency of nutrient absorption and energy storage.

Characterization of the core elements of the NF-κB signaling pathway of the sea anemone Nematostella vectensis.

The sea anemone Nematostella vectensis is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish, and corals. In insects and vertebrates, the NF-κB pathway is required for cellular and organismal responses to various stresses, including pathogens and chemicals, as well as for several developmental processes. Herein, we have characterized proteins that comprise the core NF-κB pathway in Nematostella, including homologs of NF-κB, IκB, Bcl-3, and IκB kinase (IKK). We show that N. vectensis NF-κB (Nv-NF-κB) can bind to κB sites and activate transcription of reporter genes containing multimeric κB sites or the Nv-IκB promoter. Both Nv-IκB and Nv-Bcl-3 interact with Nv-NF-κB and block its ability to activate reporter gene expression. Nv-IKK is most similar to human IKKε/TBK kinases and, in vitro, can phosphorylate Ser47 of Nv-IκB. Nv-NF-κB is expressed in a subset of ectodermal cells in juvenile and adult Nematostella anemones. A bioinformatic analysis suggests that homologs of many mammalian NF-κB target genes are targets for Nv-NF-κB, including genes involved in apoptosis and responses to organic compounds and endogenous stimuli. These results indicate that NF-κB pathway proteins in Nematostella are similar to their vertebrate homologs, and these results also provide a framework for understanding the evolutionary origins of NF-κB signaling.

The miR-15/107 group of microRNA genes: evolutionary biology, cellular functions, and roles in human diseases.

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined based on sequence similarity near the mature miRNAs' 5' end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete; thus, a definitive taxonomic classification (e.g., designation as a "superfamily") is currently not possible. While all vertebrates studied to date express miR-15a, miR-15b, miR-16, miR-103, and miR-107, mammals alone are known to express miR-195, miR-424, miR-497, miR-503, and miR-646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate to high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray matter and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103 and also support the importance of the mature miRNAs' 5' seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically oriented translational researchers alike.

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.

Two alleles of NF-kappaB in the sea anemone Nematostella vectensis are widely dispersed in nature and encode proteins with distinct activities.

BACKGROUND: NF-kappaB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-kappaB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain. METHODS/PRINCIPAL FINDINGS: We characterized two NF-kappaB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-kappaB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-kappaB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-kappaB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-kappaB proteins affect DNA binding as in human NF-kappaB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant. CONCLUSIONS/SIGNIFICANCE: These results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-kappaB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.

Domain duplication, divergence, and loss events in vertebrate Msx paralogs reveal phylogenomically informed disease markers.

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. RESULTS: Vertebrate Msx sequences sort into distinct Msx1, Msx2 and Msx3 clades. The sister-group relationship between MSX1 and MSX2 reflects their derivation from the 4p/5q chromosomal paralogon, a derivative of the original "MetaHox" cluster. We demonstrate physical linkage between Msx and other MetaHox genes (Hmx, NK1, Emx) in a cnidarian. Seven conserved domains, including two Groucho repression domains (N- and C-terminal), were present in the ancestral Msx. In cnidarians, the Groucho domains are highly similar. In vertebrate Msx1, the N-terminal Groucho domain is conserved, while the C-terminal domain diverged substantially, implying a novel function. In vertebrate Msx2 and Msx3, the C-terminal domain was lost. MSX1 mutations associated with ectodermal dysplasia or orofacial clefting disorders map to conserved domains in a non-random fashion. CONCLUSION: Msx originated from a MetaHox ancestor that also gave rise to Tlx, Demox, NK, and possibly EHGbox, Hox and ParaHox genes. Duplication, divergence or loss of domains played a central role in the functional evolution of Msx. Duplicated domains allow pleiotropically expressed proteins to evolve new functions without disrupting existing interaction networks. Human missense sequence variants reside within evolutionarily conserved domains, likely disrupting protein function. This phylogenomic evaluation of candidate disease markers will inform clinical and functional studies.

Comparative anatomy and histology of developmental and parasitic stages in the life cycle of the lined sea anemone Edwardsiella lineata.

The evolution of parasitism is often accompanied by profound changes to the developmental program. However, relatively few studies have directly examined the developmental evolution of parasitic species from free-living ancestors. The lined sea anemone Edwardsiella lineata is a relatively recently evolved parasite for which closely related free-living outgroups are known, including the starlet sea anemone Nematostella vectensis. The larva of E. lineata parasitizes the ctenophore Mnemiopsis leidyi, and, once embedded in its host, the anemone assumes a novel vermiform body plan. That we might begin to understand how the developmental program of this species has been transformed during the evolution of parasitism, we characterized the gross anatomy, histology, and cnidom of the parasitic stage, post-parasitic larval stage, and adult stage of the E. lineata life cycle. The distinct parasitic stage of the life cycle differs from the post-parasitic larva with respect to overall shape, external ciliation, cnida frequency, and tissue architecture. The parasitic stage and planula both contain holotrichs, a type of cnida not previously reported in Edwardsiidae. The internal morphology of the post-parasitic planula is extremely similar to the adult morphology, with a complete set of mesenterial tissue and musculature despite this stage having little external differentiation. Finally, we observed 2 previously undocumented aspects of asexual reproduction in E. lineata: (1) the parasitic stage undergoes transverse fission via physal pinching, the first report of asexual reproduction in a pre-adult stage in the Edwardsiidae; and (2) the juvenile polyp undergoes transverse fission via polarity reversal, the first time this form of fission has been reported in E. lineata.

The evolutionary origin of the Runx/CBFbeta transcription factors--studies of the most basal metazoans.

BACKGROUND: Members of the Runx family of transcriptional regulators, which bind DNA as heterodimers with CBFbeta, are known to play critical roles in embryonic development in many triploblastic animals such as mammals and insects. They are known to regulate basic developmental processes such as cell fate determination and cellular potency in multiple stem-cell types, including the sensory nerve cell progenitors of ganglia in mammals. RESULTS: In this study, we detect and characterize the hitherto unexplored Runx/CBFbeta genes of cnidarians and sponges, two basal animal lineages that are well known for their extensive regenerative capacity. Comparative structural modeling indicates that the Runx-CBFbeta-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFbeta dimerization in either of the proteins mirrored by compensatory changes in the binding partner. In situ hybridization studies reveal that Nematostella Runx and CBFbeta are expressed predominantly in small isolated foci at the base of the ectoderm of the tentacles in adult animals, possibly representing neurons or their progenitors. CONCLUSION: These results reveal that Runx and CBFbeta likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFbeta-DNA complex has remained extremely conserved since the human-sponge divergence. The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

Intron retention as a posttranscriptional regulatory mechanism of neurotoxin expression at early life stages of the starlet anemone Nematostella vectensis.

Sea anemones use an arsenal of peptide neurotoxins accumulated in special stinging cells (nematocytes) for defense and predation. Intriguingly, genomic analysis of Nematostella vectensis revealed only a single toxin, Nv1 (N. vectensis toxin 1), encoded by multiple extremely conserved genes. We examined the toxic potential of Nv1 and whether it is produced by the three developmental stages (embryo, planula, and polyp) of Nematostella. Nv1 was expressed in recombinant form and, similarly to Type I sea anemone toxins, inhibited the inactivation of voltage-gated sodium channels. However, in contrast to the other toxins, Nv1 revealed high specificity for insect over mammalian voltage-gated sodium channels. Transcript analysis indicated that multiple Nv1 loci are transcribed at all developmental stages of N. vectensis, whereas splicing of these transcripts is restricted to the polyp stage. This finding suggests that regulation of Nv1 synthesis is posttranscriptional and that the embryo and planula stages do not produce the Nv1 toxin. This rare phenomenon of intron retention at the early developmental stages is intriguing and raises the question as to the mechanism enabling such differential expression in sea anemones.

Rel homology domain-containing transcription factors in the cnidarian Nematostella vectensis.

The Rel/NF-kappaB and NFAT families of transcription factors are related through an N-terminal DNA-binding domain called the Rel Homology domain (RHD). Neither the RHD nor the NF-kappaB pathway has been identified in a basal (i.e., nonbilaterian) animal phylum. Using genomic and cDNA databases, we have identified two RHD domain-containing proteins from the cnidarian Nematostella vectensis: an NF-kappaB-like protein (Nv-NF-kappaB) and an NFAT-like protein (Nv-NFAT). The gene structure and RHD predicted amino acid sequence of Nv-nfkb are similar to those of the vertebrate NF-kappaB p50/p52 proteins, whereas the sequence of Nv-NFAT allows only ambiguous assignment to the NFAT family. Nv-NF-kappaB lacks the C-terminal IkappaB-like sequences present in all other NF-kappaB proteins. There are, however, two IkappaB-like genes in Nematostella encoded by loci distinct from Nv-nfkb. The separate nfkb and ikb genes of Nematostella may reflect the ancestral metazoan condition, suggesting that a gene fusion event created the nfkb genes in Drosophila and vertebrates. Nematostella also has genes that encode upstream and downstream components of the vertebrate NF-kappaB signaling pathway. Upstream components include Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase. Downstream components include the NF-kappaB coactivator protein Bcl-3 and several NF-kappaB target genes. These results demonstrate that RHD-containing transcription factors and associated pathways are evolutionarily more ancient than previously known. Moreover, they suggest models for the evolutionary diversification of the insect and vertebrate Rel/NF-kappaB/IkappaB and NFAT gene families and suggest that cnidarians possess an NF-kappaB-regulated developmental or stress response pathway.

Qualitative shift to indirect development in the parasitic sea anemone Edwardsiella lineata.

Direct development lies at 1 end of a continuum that encompasses various degrees of indirect development. Indirect development exists where a larval stage is interposed between the embryo and the adult and undergoes metamorphosis, though the ecological and morphological distinctiveness of the larval stage relative to the adult stage can vary tremendously. There are numerous empirical examples where direct development has evolved from indirect development, but little empirical evidence describing a recent transition from direct to indirect development. Here, we suggest 4 criteria for defining indirect, and therefore metamorphic, life histories. We then apply these criteria to address the planula-polyp transition in cnidarians, focusing on 2 species in the anthozoan family Edwardsiidae. The lined sea anemone, Edwardsiella lineata, has made a qualitative shift towards indirect development that coincides with, and was potentially facilitated by, the evolution of endoparasitism. We compare E. lineata's development with that of a closely related sea anemone, Nematostella vectensis, where the nonfeeding planula gradually develops the morphology of the adult polyp. In E. lineata, a novel parasitic life history stage is interposed between the planula and the polyp. We discuss how the evolution of endoparasitism could facilitate the evolution metamorphic life histories.

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.

Protein evolution: structure-function relationships of the oncogene beta-catenin in the evolution of multicellular animals.

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the 'natural experiments' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.