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The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERß). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERß PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERß, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERß.
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In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERß:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERß binding to ER-specific DNA response elements (EREs), ERß-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERß-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERß as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERß and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERß activity, validating the GFP-ERß:PRL-HeLa assay as a screening tool for potential ERß active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERß:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERß EDCs and improve upon currently available screening tools for this understudied nuclear receptor.
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During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear whether npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here, we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Células Endoteliais , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/genéticaRESUMO
In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for the identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative concentration-response information on estrogenic activity of chemical compounds.
Assuntos
Disruptores Endócrinos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Estrogênios , Genes Reporter , Peixe-Zebra/genéticaRESUMO
Steroid hormones bind receptors in the cell nucleus and in the cell membrane. The most widely studied class of steroid hormone receptors are the nuclear receptors, named for their function as ligand-dependent transcription factors in the cell nucleus. Nuclear receptors, such as estrogen receptor alpha, can also be anchored to the plasma membrane, where they respond to steroids by activating signaling pathways independent of their function as transcription factors. Steroids can also bind integral membrane proteins, such as the G protein-coupled estrogen receptor. Membrane estrogen and progestin receptors have been cloned and characterized in vitro and influence the development and function of many organ systems. Membrane androgen receptors were cloned and characterized in vitro, but their function as androgen receptors in vivo is unresolved. We review the identity and function of membrane proteins that bind estrogens, progestins, and androgens. We discuss evidence that membrane glucocorticoid and mineralocorticoid receptors exist, and whether glucocorticoid and mineralocorticoid nuclear receptors act at the cell membrane. In many cases, integral membrane steroid receptors act independently of nuclear steroid receptors, even though they may share a ligand.
Assuntos
Hormônios Esteroides Gonadais/metabolismo , Receptores de Esteroides/metabolismo , Animais , Membrana Celular/metabolismo , HumanosRESUMO
BACKGROUND AND AIMS: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions. APPROACH AND RESULTS: Exposure of zebrafish embryos to 17ß-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b-/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development. CONCLUSIONS: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.
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Sistema Biliar/embriologia , Estradiol/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fígado/embriologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistema Biliar/citologia , Sistema Biliar/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Embrião não Mamífero , Estradiol/administração & dosagem , Receptor beta de Estrogênio/genética , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/fisiologia , Fígado/citologia , Fígado/metabolismo , Masculino , Modelos Animais , Morfolinos/administração & dosagem , Morfolinos/genética , Transdução de Sinais/genética , Células-Tronco/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
OBJECTIVE: Maternal folate (vitamin B9) status is the largest known modifier of neural tube defect risk, so we evaluated folate-related mechanisms of action for dolutegravir (DTG) developmental toxicity. DESIGN: Folate receptor 1 (FOLR1) was examined as a target for DTG developmental toxicity using protein and cellular interaction studies and an animal model. METHODS: FOLR1 competitive binding studies were used to test DTG for FOLR1 antagonism. Human placenta cell line studies were used to test interactions with DTG, folate, and cations. Zebrafish were selected as an animal model to examine DTG-induced developmental toxicity and rescue strategies. RESULTS: FOLR1 binding studies indicate DTG is a noncompetitive FOLR1 antagonist at therapeutic concentrations. In-vitro testing indicates calcium (2âmmol/l) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase. Early embryonic exposure to DTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity. CONCLUSION: Folates and FOLR1 are established modifiers of risk for neural tube defects, and binding data indicates DTG is a partial antagonist of FOLR1. Supplemental folate can ameliorate increased developmental toxicity due to DTG in zebrafish. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based antiretroviral therapy in women of childbearing age.
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Receptor 1 de Folato/antagonistas & inibidores , Ácido Fólico/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/toxicidade , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Suplementos Nutricionais , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Receptor 1 de Folato/genética , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/toxicidade , Humanos , Modelos Animais , Oxazinas , Piperazinas , Gravidez , Piridonas , Testes de Toxicidade , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxicants and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have 3 paralogs of AHR: ahr1a, ahr1b, and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a;ahr1b single and double mutant zebrafish. We found that ahr1a;ahr1b single and double mutants were morphologically normal whereas ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a;ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. Next, we analyzed the function of zebrafish AHRs activated by environmental ligands. The prototypical AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces toxicity in humans and rodents via AHR and causes cardiotoxicity in zebrafish embryos. It has been shown that embryos with mutations in ahr2 are resistant to TCDD toxicity, yet it is unclear whether ahr1 receptors are required. Furthermore, though AHR was shown to interact with estrogen receptor alpha following TCDD treatment, it is not known whether this interaction is constitutive or context-dependent. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a;ahr1b or estrogen receptor genes are susceptible to TCDD toxicity whereas ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.
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Nadadeiras de Animais/crescimento & desenvolvimento , Cardiotoxicidade/etiologia , Anormalidades Craniofaciais/genética , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/metabolismo , Crânio/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/efeitos dos fármacos , Animais , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Anormalidades Craniofaciais/metabolismo , Embrião não Mamífero , Feminino , Masculino , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Crânio/efeitos dos fármacos , Crânio/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-ZebraRESUMO
BACKGROUND & AIMS: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. METHODS: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. RESULTS: Exposure to 17ß-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. CONCLUSIONS: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. TRANSCRIPT PROFILING: The accession number in the Gene Expression Omnibus is GSE92544.
Assuntos
Carcinoma Hepatocelular/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Neoplasias Hepáticas/metabolismo , Fígado/crescimento & desenvolvimento , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Hepatócitos , Humanos , Fígado/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/patologia , Regeneração Hepática , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Receptores Acoplados a Proteínas G/genética , Fatores Sexuais , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Carga Tumoral/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Estrogens regulate vertebrate development and function through binding to nuclear estrogen receptors α and ß (ERα and ERß) and the G protein-coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERß are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo, we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function was normal in gper mutant ovaries compared with wild type. Our findings suggest that GPER is not required for sex determination, ovary development, or fertility in zebrafish.
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Ovário/metabolismo , Receptores Acoplados a Proteínas G/genética , Análise para Determinação do Sexo/métodos , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Masculino , Mutação , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Razão de Masculinidade , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
In 2005, two groups independently discovered that the G protein-coupled receptor GPR30 binds estradiol in cultured cells and, in response, initiates intracellular signaling cascades Revankar et al. (2005), Thomas et al. (2005). GPR30 is now referred to as GPER, the G-protein coupled estrogen receptor Prossnitz and Arterburn (2015). While studies in animal models are illuminating GPER function, there is controversy as to whether GPER acts as an autonomous estrogen receptor in vivo, or whether GPER interacts with nuclear estrogen receptor signaling pathways in response to estrogens. Here, we review the evidence that GPER acts as an autonomous estrogen receptor in vivo and discuss experimental approaches to test this hypothesis directly. We propose that the degree to which GPER influences nuclear estrogen receptor signaling likely depends on cell type, developmental stage and pathology.
Assuntos
Receptor Cross-Talk/fisiologia , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Núcleo Celular/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Humanos , Transdução de Sinais/fisiologiaRESUMO
To study the effects of environmental endocrine disruptor compounds (EDCs) on aquatic animals, embryos and larvae are typically incubated in water containing defined concentrations of EDCs. However, the amount of EDC uptake into the animal is often difficult to determine. Using radiolabeled estradiol ([3H]E2), we previously developed a rapid, straightforward assay to measure estradiol uptake from water into zebrafish embryos and larvae. Here, we extend this approach to measure the uptake of two additional EDCs, bisphenol A (BPA) and ethinyl estradiol (EE2). As with E2, the uptake of each compound by individual larvae was low (<6%), and increased with increasing concentration, duration, and developmental stage. We found that E2 and EE2 had similar uptake under equivalent exposure conditions, while BPA had comparatively lower uptake. One application of this assay is to test factors that influence EDC uptake or efflux. It has been suggested that persistent organic pollutants (POPs) inhibit ABC transporters that may normally efflux EDCs and their metabolites, inducing toxicity in aquatic organisms. We measured [3H]E2 levels in zebrafish in the presence or absence of the POP PDBE-100, and cyclosporine A, a known inhibitor of ABC transporters. Neither chemical significantly affected [3H]E2 levels in zebrafish, suggesting that zebrafish maintain estradiol efflux in the presence of PDBE-100, independently of cyclosporine A-responsive transporters. These uptake results will be a valuable reference for EDC exposure studies in developing zebrafish, and provide a rapid assay to screen for chemicals that influence estrogen-like EDC levels in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Embrião não Mamífero/metabolismo , Disruptores Endócrinos/metabolismo , Poluentes Químicos da Água/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Fatores Etários , Animais , Compostos Benzidrílicos/metabolismo , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Disruptores Endócrinos/toxicidade , Estradiol/metabolismo , Etinilestradiol/metabolismo , Éteres Difenil Halogenados/farmacologia , Larva/metabolismo , Fenóis/metabolismo , Fatores de Tempo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERß), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERß mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERß mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.
Assuntos
Embrião não Mamífero/fisiologia , Estradiol/administração & dosagem , Frequência Cardíaca/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Estrogênios/análise , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mutação , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Tri-Iodotironina/análise , Proteínas de Peixe-Zebra/genéticaRESUMO
Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.
Assuntos
Embrião não Mamífero/metabolismo , Estradiol/metabolismo , Larva/metabolismo , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Larva/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds.
Assuntos
Disruptores Endócrinos/farmacologia , Estrogênios/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Receptores de Estrogênio/efeitos dos fármacos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Microscopia de Fluorescência , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fluxo de Trabalho , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.
Assuntos
Proteínas de Fluorescência Verde/química , Microscopia de Fluorescência/métodos , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Automação , Embrião não Mamífero , Feminino , Ligantes , Masculino , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismoRESUMO
Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.
Assuntos
Antagonistas de Estrogênios/farmacologia , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Compostos Benzidrílicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Efrina-B2/antagonistas & inibidores , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Fulvestranto , Genisteína/farmacologia , Resposta ao Choque Térmico , Morfolinos/genética , Fenóis/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Estradiol/genética , Receptores Notch/biossíntese , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
BACKGROUND: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart. METHODS: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization. RESULTS: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERß2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver. CONCLUSION: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.
Assuntos
Estrogênios/toxicidade , Animais , Animais Geneticamente Modificados , Disruptores Endócrinos/toxicidade , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismo , Peixe-ZebraRESUMO
Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17ß-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Análise por Conglomerados , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Ontologia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Masculino , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Estrogens regulate a diverse range of physiological processes and affect multiple tissues. Estrogen receptors (ERs) regulate transcription by binding to DNA at conserved estrogen response elements, and such elements have been used to report ER activity in cultured cells and in transgenic mice. We generated stable, transgenic zebrafish containing five consecutive elements upstream of a c-fos minimal promoter and green fluorescent protein (GFP) to visualize and quantify transcriptional activation in live larvae. Transgenic larvae show robust, dose-dependent estrogen-dependent fluorescent labeling in the liver, consistent with er gene expression, whereas ER antagonists inhibit GFP expression. The nonestrogenic steroids dexamethasone and progesterone fail to activate GFP, confirming ER selectivity. Natural and synthetic estrogens activated the transgene with varying potency, and two chemicals, genistein and bisphenol A, preferentially induce GFP expression in the heart. In adult fish, fluorescence was observed in estrogenic tissues such as the liver, ovary, pituitary gland, and brain. Individual estrogen-responsive neurons and their projections were visualized in the adult brain, and GFP-positive neurons increased in number after 17ß-estradiol exposure. The transgenic estrogen-responsive zebrafish allow ER signaling to be monitored visually and serve as in vivo sentinels for detection of estrogenic compounds.