Adaptive Significance of ERα Splice Variants in Killifish (Fundulus heteroclitus) Resident in an Estrogenic Environment.

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERα) mRNA but are hyporesponsive to estradiol. Analysis of ERα mRNAs in the two populations revealed differences in splicing of the gene encoding ERα (esr1). Here we tested the transactivation functions of four differentially expressed ERα mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERαΔ6 and ERαΔ6-8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERαΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERαL) and short (ERαS) 5'-variants were similar transcriptionally but differed in estrogen responsiveness (ERαS ≫ ERαL). ERαS accounted for high total ERα expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERαΔ6 and ERαΔ6-8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERα pre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.

Cloning of multiple ERα mRNA variants in killifish (Fundulus heteroclitus), and differential expression by tissue type, stage of reproduction, and estrogen exposure in fish from polluted and unpolluted environments.

To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ERα) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified 19 ERα variants, each with deletions of one or more coding exons. Four of the variants with potential functional relevance were analyzed by qPCR to test for population differences in expression by tissue type, site, sex, seasonal reproductive status and estrogen treatment. Significantly, a 5'-truncated short form variant (ERαS) was highly expressed in liver and ovary, and was associated with seasonal reproductive activity in SC but not NBH fish. Both ERαS and the full-length long variant (ERαL) were estrogen-inducible (ERαS>ERαL) but the induction response was lower in NBH than in SC fish. In contrast, NBH killifish were hyper-responsive to estrogen as measured by expression of two other estrogen responsive genes: vitellogenin (Vtg) and aromatase B (AroB). Most strikingly, two ERα deletion variants (Δ6 and Δ6-8), lacking ligand binding and activation function domains, were identified in a subset of NBH fish, where they were associated with reduced responsiveness to estrogen treatment. Together, these results support the hypothesis that alternative splicing of the esr1 gene of killifish could be an autoregulatory mechanism by which estrogen modulates the differential expression of ERα, and suggests a novel and adaptive mechanistic response to xenoestrogenic exposure.

Multiple structurally distinct ERα mRNA variants in zebrafish are differentially expressed by tissue type, stage of development and estrogen exposure.

It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ERs) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαA(L)) and short (ERα(S)) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERα(L) mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERα(S) mRNA was preferentially expressed in liver (female>male) and ovary. Neither ERα(L) nor ERα(S) transcripts varied significantly during development, but 17ß-estradiol selectively increased accumulation of ERα(S) mRNA (∼170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERα(S)-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERα(S) in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms.

Morpholino-mediated knockdown of ERα, ERßa, and ERßb mRNAs in zebrafish (Danio rerio) embryos reveals differential regulation of estrogen-inducible genes.

Genetically distinct estrogen receptor (ER) subtypes (ERα and ERß) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERßa and ERßb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERßa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERßb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERßa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.

Time Course Analysis of Gene Expression Patterns in Zebrafish Eye During Optic Nerve Regeneration.

It is well-established that neurons in the adult mammalian central nervous system (CNS) are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in approximately 21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after ONX can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. To define different phases of regeneration after ONX, alpha tubulin 1 (tuba1) and growth-associated protein 43 (gap43), markers previously shown to correspond to morphophological events, were measured by real time quantitative PCR (qPCR). Microarray analysis was then performed at defined intervals (6 hours, 1, 4, 12, and 21 days) post-ONX and compared to SHAM. Results show that optic nerve damage induces multiple, phase-related transcriptional programs, with the maximum number of genes changed and highest fold-change occurring at 4 days. Several functional groups affected by optic nerve regeneration, including cell adhesion, apoptosis, cell cycle, energy metabolism, ion channel activity, and calcium signaling, were identified. Utilizing the whole eye allowed us to identify signaling contributions from the vitreous, immune and glial cells as well as the neural cells of the retina. Comparisons between our dataset and transcriptional profiles from other models of regeneration in zebrafish retina, heart and fin revealed a subset of commonly regulated transcripts, indicating shared mechanisms in different regenerating tissues. Knowledge of gene expression patterns in all components of the eye in a model of successful regeneration provides an entry point for functional analyses, and will help in devising hypotheses for testing normal and toxic regulatory factors.

Estrogen responses in killifish (Fundulus heteroclitus) from polluted and unpolluted environments are site- and gene-specific.

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 (ER alpha). In contrast to our previous observation of elevated ER alpha 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 ER alpha) and less sensitive (higher EC(50) 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 ER alpha mRNA in several tissues from adult NBH fish vs SC fish (Greytak and Callard, 2007), we predicted estrogen hyporesponsiveness; however, induction of ER alpha 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 ER alpha 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 life-stage-specific.

Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.

BACKGROUND: Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. RESULTS: QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 - 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 - 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. CONCLUSION: This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Cloning of three estrogen receptors (ER) from killifish (Fundulus heteroclitus): differences in populations from polluted and reference environments.

Epidemiological, wildlife, and laboratory studies support the hypothesis that chemicals released into the environment through anthropogenic activities are responsible for abnormalities of reproduction and development. Although the New Bedford Harbor (NBH) killifish population has survived and reproduced successfully for >50 yr ( approximately 20 generations), fish have high body burdens of the major NBH contaminants (polychlorinated biphenyls); elevated levels of P450 aromatase B and vitellogenin mRNA (markers of estrogen effect); and evidence of endocrine disruption. To investigate possible adaptive changes in the estrogen response system of NBH killifish, we cloned the estrogen receptors (ER) from killifish populations resident in NBH and a relatively unpolluted reference site (Scorton Creek MA, SC). ERalpha, -betaa, and -betab cDNAs encoding full-length polypeptides of 620, 543, and 672 amino acids, respectively, were identified. Each ER subtype had multiple splice variants, single nucleotide polymorphisms (SNPs), and a characteristic tissue distribution and developmental profile. As measured by real-time quantitative RT-PCR analysis, the overall tissue distribution of each ER was similar in NBH and SC fish, implying that the regulatory pathways which maintain tissue-specific expression are largely unchanged by long term pollutant exposure. Nonetheless, a striking difference was seen in the quantity of mRNA of the estrogen-inducible gene ERalpha, which was significantly lower in brain, liver and ovaries of reproductively active NBH as compared to SC females. Paradoxically, despite the "estrogenic" NBH environment, ERalpha mRNA levels did not differ in reproductively inactive NBH and SC females, or in males at the two sites at any time of year. We interpret results in NBH fish as due in part to a deficit of circulating estrogen, and in part to pollutant-mediated hyporesponsiveness of the ERalpha gene. In marked contrast to adult fish, ERalpha was elevated approximately 5-fold in NBH as compared to SC embryos/larvae, perhaps indicative of estrogenic chemicals in yolk. We conclude that contaminants in the NBH environment impact molecular components of the estrogen signaling pathways in resident killifish populations. Whether these changes are transient or heritable requires further study.

Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation.

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female>male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.

Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments.

Populations of killifish (Fundulus heteroclitus) persist in many different highly polluted environment indicative of adaptation or tolerance. In this study, we sought to determine whether long term, multigenerational exposures to environmental contaminants has affected reproductively relevant genes and biological processes. A homology cloning strategy was used to isolate the killifish cytochrome P450 aromatase (P450arom, estrogen synthetase) cDNAs. Consistent with previous fish studies, killifish were found to have two P450arom forms, which segregated into A- and B-gene clades and were differentially expressed in brain (B >> A) and ovary (A >> B). Comparison of killifish from highly polluted (New Bedford Harbor, NBH) and unpolluted (Scorton Creek, SC) environments revealed no site-related differences in P450arom coding sequences or in overall tissue distribution patterns. As measured by real-time quantitative PCR (QPCR) analysis, however, P450arormB (a known marker of estrogen effect) was approximately two-fold higher in the brain of NBH than of SC fish, a difference seen in reproductively active and inactive males and females. Providing further evidence of exposure to estrogen-like pollutants or metabolites in NBH, vitellogenin (vtg) mRNA and protein were elevated in seasonally active and inactive males, and in reproductively inactive females, when compared to SC fish. By contrast, during the period of reproductive activity, NBH females had a lower gonadosomatic index, lower plasma estrogen, a decreased hepatosomatic index, and reduced vtg expression as compared to SC females, indicating that the female hypothalamic-pituitary-gonadal (HPG)-liver axis is impaired in the polluted environment. As measured by a decrease in plasma androgen (but not GSI), the male HPG axis was impaired in reproductively active NBH versus SC fish. In agreement with reports that NBH killifish are resistant to dioxin-like chemicals (DLC) that activate arylhydrocarbon receptor (AhR) signaling, ovarian P450aromA (a marker of dioxin-like effect in zebrafish embryos) did not differ in SC and NBH fish. In conclusion, the killifish population at the NBH Superfund site maintains a level of reproductive competence in the face of evidence of exposure to estrogen-like pollutants and endocrine disruption.