The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were origina... more The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to the estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the diversity, function, and regulation of ERRs in non-mammalian species are not well understood. In this study, we report the cloning of four ERR cDNAs from the Atlantic killifish, Fundulus heteroclitus, along with adult tissue expression and estrogen responsiveness. Phylogenetic analysis indicates that F. heteroclitus (Fh)ERRa is an ortholog of the single ERRa identified in mammals, pufferfish, and zebrafish. FhERRba and FhERRbb are co-orthologs of the mammalian ERRb. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRgb, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRa was broadly expressed. FhERRba was expressed at apparently low levels in eye, brain, and ovary. FhERRbb was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRgb was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRba and FhERRbb are consistent with subfunctionalization of the ERRb paralogs. Induction of ERRa mRNA by exogenous estrogen exposure has been reported in some mammalian tissues. In adult male killifish, ERR expression did not significantly change following estradiol injection, but showed a trend toward a slight induction (three-to five-fold) of ERRa expression in heart. In a second, more targeted experiment, expression of ERRa in adult female killifish was downregulated 2$5-fold in the heart following estradiol injection. In summary, our results indicate that killifish contain additional ERR genes relative to mammals, including ERRb paralogs. In addition, regulation of ERRa expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.
Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine d... more Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ERα). In contrast to our previous observation of elevated ERα in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ERα) and less sensitive (higher EC50 for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ERα mRNA in several tissues from adult NBH fish vs SC fish , we predicted estrogen hyporesponsiveness; however, induction of ERα by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ERα mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type-and lifestage specific.
The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has ... more The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR 715 ) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRR⌬8) that lacks exon 8 of AHRR 715 . AHRR⌬8 was the predominant AHRR form expressed in human tissues and cell lines. AHRR⌬8 effectively repressed AHR-dependent transactivation, whereas AHRR 715 was much less active. Similarly, AHRR⌬8, but not AHRR 715 , formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRR⌬8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRR⌬8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRR⌬8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor ␣ but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRR⌬8-Pro 185 and -Ala 185 variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRR⌬8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.
Sea surface temperature (SST) across much of the tropics has increased by 0.4° to 1°C since the m... more Sea surface temperature (SST) across much of the tropics has increased by 0.4° to 1°C since the mid-1970s. A parallel increase in the frequency and extent of coral bleaching and mortality has fueled concern that climate change poses a major threat to the survival of coral reef ecosystems worldwide. Here we show that steadily rising SSTs, not ocean acidification, are already driving dramatic changes in the growth of an important reef-building coral in the central Red Sea. Three-dimensional computed tomography analyses of the massive coral Diploastrea heliopora reveal that skeletal growth of apparently healthy colonies has declined by 30% since 1998. The same corals responded to a short-lived warm event in 1941/1942, but recovered within 3 years as the ocean cooled. Combining our data with climate model simulations by the Intergovernmental Panel on Climate Change, we predict that should the current warming trend continue, this coral could cease growing altogether by 2070.
To survive long periods of low food availability, some calanoid copepods have a life history that... more To survive long periods of low food availability, some calanoid copepods have a life history that includes a diapause phase during which copepodids delay development to adulthood, migrate to depth, reduce metabolism, and utilize stored lipids for nourishment. While seasonal patterns in diapause have been described, the environmental and physiological regulation of diapause has not been elucidated. We collected Calanus finmarchicus C5 copepodids from surface (0 to 39 m) and deep (157 to 201 m) waters in the Gulf of Maine, and both morphological and biochemical measurements indicated that these copepodids were from active and diapausing populations, respectively. Two complementary molecular techniques were used to compare gene expression in these 2 groups: (1) suppressive subtractive hybridization (SSH) was used to identify genes that may be differentially expressed, and (2) quantitative real-time RT-PCR was used to characterize patterns of gene expression in individual copepodids. Three genes associated with lipid synthesis, transport and storage (ELOV, FABP, RDH) were upregulated (more highly expressed) in active copepods, particularly those with small oil sacs. Expression of ferritin was greater in diapausing copepods with large oil sacs, consistent with a role of ferritin in chelating metals to protect cells from oxidative stress and/or delay development. Ecdysteroid receptor (EcR) expression was greater in diapausing copepods, highlighting the need for further investigation into endocrine regulation of copepod development. This study represents the first molecular characterization of gene expression associated with calanoid copepod diapause and provides a foundation for future investigations of the underlying mechanisms that regulate diapause.
Epizootic shell disease is a poorly understood condition that has significantly affected the Amer... more Epizootic shell disease is a poorly understood condition that has significantly affected the American lobster fishery in New England (northeastern US) since the 1990s. Here we present the results of a study to identify changes in gene expression in lobsters exhibiting symptoms of epizootic shell disease. Suppressive subtractive hybridization (SSH) was used to compare gene expression between cDNA pools from diseased (symptomatic) and apparently healthy (asymptomatic) lobsters. Subsequently, quantitative real-time polymerase chain reaction (qPCR) was used to measure expression of nine genes that were differentially-expressed in the SSH analysis, in seven tissues (muscle, gill, heart, hepatopancreas, brain, branchiostegite, gonad) dissected from individual symptomatic and asymptomatic lobsters. Expression of arginine kinase (involved in cellular energetics) was significantly decreased in muscle of symptomatic lobsters. Expression of hemocyanin (a respiratory hemolymph protein involved in oxygen transport) was highest in hepatopancreas and showed highly variable expression with a trend toward higher expression in asymptomatic individuals. 2-Macroglobulin (involved in the innate immune system) was most highly expressed in the ovary, particularly of symptomatic lobsters. The ESTs produced through this study add to the fledgling field of crustacean genomics and revealed three genes that could be further evaluated in lobsters of varying shell disease severity, molt stage, and reproductive condition, for possible implication in epizootic shell disease.
Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological process... more Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.
In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone r... more In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.
Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnid... more Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnidarians contain a diverse complement of steroids, sterols, and other lipid metabolites, relatively little is known of the endogenous steroid metabolism or function in cnidarian tissues. Incubations of cnidarian tissues with steroid substrates have indicated the presence of steroid metabolizing enzymes, particularly enzymes with 17-hydroxysteroid dehydrogenase (17-HSD) activity. Through analysis of the genome of the starlet sea anemone, Nematostella vectensis, we identified a suite of genes in the short chain dehydrogenase/reductase (SDR) superfamily including homologs of genes that metabolize steroids in other animals. A more detailed analysis of Hsd17b4 revealed complex evolutionary relationships, apparent intron loss in several taxa, and predominantly adult expression in N. vectensis. Due to its ease of culture and available molecular tools N. vectensis is an excellent model for investigation of cnidarian steroid metabolism and gene function.
Background: Circadian rhythms in behavior and physiology are the observable phenotypes from cycle... more Background: Circadian rhythms in behavior and physiology are the observable phenotypes from cycles in expression of, interactions between, and degradation of the underlying molecular components. In bilaterian animals, the core molecular components include Timeless-Timeout, photoreceptive cryptochromes, and several members of the basic-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. While many of core circadian genes are conserved throughout the Bilateria, their specific roles vary among species. Here, we identify and experimentally study the rhythmic gene expression of conserved circadian clock members in a sea anemone in order to characterize this gene network in a member of the phylum Cnidaria and to infer critical components of the clockwork used in the last common ancestor of cnidarians and bilaterians.
Background: Nuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors ... more Background: Nuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors that play critical roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes), comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined NR expression during ctenophore development. Results: We report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the subfamily NR2A . Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD). NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor. Conclusions: Our study provides the first description of NRs from ctenophores, including the full complement from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives that cluster with only one subfamily (NR2A). Ctenophores and sponges have a similarly restricted NR complement supporting the hypothesis that the original NR was HNF4-like and that these lineages are the first two branches from the animal tree. The absence of a zinc-finger DNA-binding domain in the two ctenophore species suggests two hypotheses: this domain may have been secondarily lost within the ctenophore lineage or, if ctenophores are the first branch off the animal tree, the original NR may have lacked the canonical DBD. Phylogenomic analyses and categorization of NRs from all four early diverging animal phyla compared with the complement from bilaterians suggest the rate of NR diversification prior to the cnidarian-bilaterian split was relatively modest, with independent radiations of several NR subfamilies within the cnidarian lineage.
Background: Nuclear receptors are a superfamily of metazoan transcription factors that regulate d... more Background: Nuclear receptors are a superfamily of metazoan transcription factors that regulate diverse developmental and physiological processes. Sequenced genomes from an increasing number of bilaterians have provided a more complete picture of duplication and loss of nuclear receptors in protostomes and deuterostomes but have left open the question of which nuclear receptors were present in the cnidarian-bilaterian ancestor. In addition, nuclear receptor expression and function are largely uncharacterized within cnidarians, preventing determination of conserved and novel nuclear receptor functions in the context of animal evolution.
Conserved interactions among proteins or other molecules can provide strong evidence for coevolut... more Conserved interactions among proteins or other molecules can provide strong evidence for coevolution across their evolutionary history. Diverse phylogenetic methods have been applied to identify potential coevolutionary relationships. In most cases, these methods minimally require comparisons of orthologous sequences and appropriate controls to separate effects of selection from the overall evolutionary relationships. In vertebrates, androgen receptor (AR) and cytochrome p450 aromatase (CYP19) share an affinity for androgenic steroids, which serve as receptor ligands and enzyme substrates. In a recent study, Tiwary and Li (Tiwary BK, Li W-H. 2009. Parallel evolution between aromatase and androgen receptor in the animal kingdom. Mol Biol Evol. 26:123-129) reported that AR and CYP19 displayed a signature of ancient and conserved interactions throughout all the Eumetazoa (i.e., cnidarians, protostomes, and deuterostomes). Because these findings conflicted with a number of previous studies, we reanalyzed the data set used by Tiwary and Li. First, our analyses demonstrate that the invertebrate genes used in the previous analysis are not orthologous sequences but instead represent a diverse set of nuclear receptors and CYP enzymes with no confirmed or hypothesized relationships with androgens. Second, we show that 1) their analytical approach, which measures correlations in evolutionary distances between proteins, potentially led to spurious significant relationships due simply to conserved domains and 2) control comparisons provide positive evidence for a strong influence of evolutionary history. We discuss how corrections to this method and analysis of key taxa (e.g., duplications in the teleost fish and suiform lineages) can inform investigations of the coevolutionary relationships between AR and aromatase.
The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were origina... more The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to the estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the diversity, function, and regulation of ERRs in non-mammalian species are not well understood. In this study, we report the cloning of four ERR cDNAs from the Atlantic killifish, Fundulus heteroclitus, along with adult tissue expression and estrogen responsiveness. Phylogenetic analysis indicates that F. heteroclitus (Fh)ERRa is an ortholog of the single ERRa identified in mammals, pufferfish, and zebrafish. FhERRba and FhERRbb are co-orthologs of the mammalian ERRb. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRgb, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRa was broadly expressed. FhERRba was expressed at apparently low levels in eye, brain, and ovary. FhERRbb was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRgb was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRba and FhERRbb are consistent with subfunctionalization of the ERRb paralogs. Induction of ERRa mRNA by exogenous estrogen exposure has been reported in some mammalian tissues. In adult male killifish, ERR expression did not significantly change following estradiol injection, but showed a trend toward a slight induction (three-to five-fold) of ERRa expression in heart. In a second, more targeted experiment, expression of ERRa in adult female killifish was downregulated 2$5-fold in the heart following estradiol injection. In summary, our results indicate that killifish contain additional ERR genes relative to mammals, including ERRb paralogs. In addition, regulation of ERRa expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.
Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine d... more Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ERα). In contrast to our previous observation of elevated ERα in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ERα) and less sensitive (higher EC50 for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ERα mRNA in several tissues from adult NBH fish vs SC fish , we predicted estrogen hyporesponsiveness; however, induction of ERα by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ERα mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type-and lifestage specific.
The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has ... more The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR 715 ) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRR⌬8) that lacks exon 8 of AHRR 715 . AHRR⌬8 was the predominant AHRR form expressed in human tissues and cell lines. AHRR⌬8 effectively repressed AHR-dependent transactivation, whereas AHRR 715 was much less active. Similarly, AHRR⌬8, but not AHRR 715 , formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRR⌬8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRR⌬8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRR⌬8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor ␣ but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRR⌬8-Pro 185 and -Ala 185 variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRR⌬8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.
Sea surface temperature (SST) across much of the tropics has increased by 0.4° to 1°C since the m... more Sea surface temperature (SST) across much of the tropics has increased by 0.4° to 1°C since the mid-1970s. A parallel increase in the frequency and extent of coral bleaching and mortality has fueled concern that climate change poses a major threat to the survival of coral reef ecosystems worldwide. Here we show that steadily rising SSTs, not ocean acidification, are already driving dramatic changes in the growth of an important reef-building coral in the central Red Sea. Three-dimensional computed tomography analyses of the massive coral Diploastrea heliopora reveal that skeletal growth of apparently healthy colonies has declined by 30% since 1998. The same corals responded to a short-lived warm event in 1941/1942, but recovered within 3 years as the ocean cooled. Combining our data with climate model simulations by the Intergovernmental Panel on Climate Change, we predict that should the current warming trend continue, this coral could cease growing altogether by 2070.
To survive long periods of low food availability, some calanoid copepods have a life history that... more To survive long periods of low food availability, some calanoid copepods have a life history that includes a diapause phase during which copepodids delay development to adulthood, migrate to depth, reduce metabolism, and utilize stored lipids for nourishment. While seasonal patterns in diapause have been described, the environmental and physiological regulation of diapause has not been elucidated. We collected Calanus finmarchicus C5 copepodids from surface (0 to 39 m) and deep (157 to 201 m) waters in the Gulf of Maine, and both morphological and biochemical measurements indicated that these copepodids were from active and diapausing populations, respectively. Two complementary molecular techniques were used to compare gene expression in these 2 groups: (1) suppressive subtractive hybridization (SSH) was used to identify genes that may be differentially expressed, and (2) quantitative real-time RT-PCR was used to characterize patterns of gene expression in individual copepodids. Three genes associated with lipid synthesis, transport and storage (ELOV, FABP, RDH) were upregulated (more highly expressed) in active copepods, particularly those with small oil sacs. Expression of ferritin was greater in diapausing copepods with large oil sacs, consistent with a role of ferritin in chelating metals to protect cells from oxidative stress and/or delay development. Ecdysteroid receptor (EcR) expression was greater in diapausing copepods, highlighting the need for further investigation into endocrine regulation of copepod development. This study represents the first molecular characterization of gene expression associated with calanoid copepod diapause and provides a foundation for future investigations of the underlying mechanisms that regulate diapause.
Epizootic shell disease is a poorly understood condition that has significantly affected the Amer... more Epizootic shell disease is a poorly understood condition that has significantly affected the American lobster fishery in New England (northeastern US) since the 1990s. Here we present the results of a study to identify changes in gene expression in lobsters exhibiting symptoms of epizootic shell disease. Suppressive subtractive hybridization (SSH) was used to compare gene expression between cDNA pools from diseased (symptomatic) and apparently healthy (asymptomatic) lobsters. Subsequently, quantitative real-time polymerase chain reaction (qPCR) was used to measure expression of nine genes that were differentially-expressed in the SSH analysis, in seven tissues (muscle, gill, heart, hepatopancreas, brain, branchiostegite, gonad) dissected from individual symptomatic and asymptomatic lobsters. Expression of arginine kinase (involved in cellular energetics) was significantly decreased in muscle of symptomatic lobsters. Expression of hemocyanin (a respiratory hemolymph protein involved in oxygen transport) was highest in hepatopancreas and showed highly variable expression with a trend toward higher expression in asymptomatic individuals. 2-Macroglobulin (involved in the innate immune system) was most highly expressed in the ovary, particularly of symptomatic lobsters. The ESTs produced through this study add to the fledgling field of crustacean genomics and revealed three genes that could be further evaluated in lobsters of varying shell disease severity, molt stage, and reproductive condition, for possible implication in epizootic shell disease.
Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological process... more Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.
In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone r... more In arthropods, ecdysteroids regulate molting by activating a heterodimer formed by the ecdysone receptor (EcR) and retinoid X receptor (RXR). While this mechanism is similar in insects and crustaceans, variation in receptor splicing, dimerization and ligand affinity adds specificity to molting processes. This study reports the EcR and RXR sequences from American lobster, a commercially and ecologically important crustacean. We cloned two EcR splice variants, both of which specifically bind ponasterone A, and two RXR variants, both of which enhance binding of ponasterone A to the EcR. Lobster EcR has high affinity for ponasterone A and muristerone and moderately high affinity for the insecticide tebufenozide. Bisphenol A, diethyl phthalate, and two polychlorinated biphenyls (PCB 29 and PCB 30), environmental chemicals shown to interfere with crustacean molting, showed little or no affinity for lobster EcR. These studies establish the molecular basis for investigation of lobster ecdysteroid signaling and signal disruption by environmental chemicals.
Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnid... more Cnidarians occupy a key evolutionary position as a sister group to bilaterian animals. While cnidarians contain a diverse complement of steroids, sterols, and other lipid metabolites, relatively little is known of the endogenous steroid metabolism or function in cnidarian tissues. Incubations of cnidarian tissues with steroid substrates have indicated the presence of steroid metabolizing enzymes, particularly enzymes with 17-hydroxysteroid dehydrogenase (17-HSD) activity. Through analysis of the genome of the starlet sea anemone, Nematostella vectensis, we identified a suite of genes in the short chain dehydrogenase/reductase (SDR) superfamily including homologs of genes that metabolize steroids in other animals. A more detailed analysis of Hsd17b4 revealed complex evolutionary relationships, apparent intron loss in several taxa, and predominantly adult expression in N. vectensis. Due to its ease of culture and available molecular tools N. vectensis is an excellent model for investigation of cnidarian steroid metabolism and gene function.
Background: Circadian rhythms in behavior and physiology are the observable phenotypes from cycle... more Background: Circadian rhythms in behavior and physiology are the observable phenotypes from cycles in expression of, interactions between, and degradation of the underlying molecular components. In bilaterian animals, the core molecular components include Timeless-Timeout, photoreceptive cryptochromes, and several members of the basic-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. While many of core circadian genes are conserved throughout the Bilateria, their specific roles vary among species. Here, we identify and experimentally study the rhythmic gene expression of conserved circadian clock members in a sea anemone in order to characterize this gene network in a member of the phylum Cnidaria and to infer critical components of the clockwork used in the last common ancestor of cnidarians and bilaterians.
Background: Nuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors ... more Background: Nuclear receptors (NRs) are an ancient superfamily of metazoan transcription factors that play critical roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes), comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined NR expression during ctenophore development. Results: We report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the subfamily NR2A . Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD). NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor. Conclusions: Our study provides the first description of NRs from ctenophores, including the full complement from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives that cluster with only one subfamily (NR2A). Ctenophores and sponges have a similarly restricted NR complement supporting the hypothesis that the original NR was HNF4-like and that these lineages are the first two branches from the animal tree. The absence of a zinc-finger DNA-binding domain in the two ctenophore species suggests two hypotheses: this domain may have been secondarily lost within the ctenophore lineage or, if ctenophores are the first branch off the animal tree, the original NR may have lacked the canonical DBD. Phylogenomic analyses and categorization of NRs from all four early diverging animal phyla compared with the complement from bilaterians suggest the rate of NR diversification prior to the cnidarian-bilaterian split was relatively modest, with independent radiations of several NR subfamilies within the cnidarian lineage.
Background: Nuclear receptors are a superfamily of metazoan transcription factors that regulate d... more Background: Nuclear receptors are a superfamily of metazoan transcription factors that regulate diverse developmental and physiological processes. Sequenced genomes from an increasing number of bilaterians have provided a more complete picture of duplication and loss of nuclear receptors in protostomes and deuterostomes but have left open the question of which nuclear receptors were present in the cnidarian-bilaterian ancestor. In addition, nuclear receptor expression and function are largely uncharacterized within cnidarians, preventing determination of conserved and novel nuclear receptor functions in the context of animal evolution.
Conserved interactions among proteins or other molecules can provide strong evidence for coevolut... more Conserved interactions among proteins or other molecules can provide strong evidence for coevolution across their evolutionary history. Diverse phylogenetic methods have been applied to identify potential coevolutionary relationships. In most cases, these methods minimally require comparisons of orthologous sequences and appropriate controls to separate effects of selection from the overall evolutionary relationships. In vertebrates, androgen receptor (AR) and cytochrome p450 aromatase (CYP19) share an affinity for androgenic steroids, which serve as receptor ligands and enzyme substrates. In a recent study, Tiwary and Li (Tiwary BK, Li W-H. 2009. Parallel evolution between aromatase and androgen receptor in the animal kingdom. Mol Biol Evol. 26:123-129) reported that AR and CYP19 displayed a signature of ancient and conserved interactions throughout all the Eumetazoa (i.e., cnidarians, protostomes, and deuterostomes). Because these findings conflicted with a number of previous studies, we reanalyzed the data set used by Tiwary and Li. First, our analyses demonstrate that the invertebrate genes used in the previous analysis are not orthologous sequences but instead represent a diverse set of nuclear receptors and CYP enzymes with no confirmed or hypothesized relationships with androgens. Second, we show that 1) their analytical approach, which measures correlations in evolutionary distances between proteins, potentially led to spurious significant relationships due simply to conserved domains and 2) control comparisons provide positive evidence for a strong influence of evolutionary history. We discuss how corrections to this method and analysis of key taxa (e.g., duplications in the teleost fish and suiform lineages) can inform investigations of the coevolutionary relationships between AR and aromatase.
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Papers by Ann Tarrant