Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental time point of 72 h post fertilization, low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after up-regulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggests a function for the transmembrane domain in liposome docking. We conclude that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

Aryl Hydrocarbon Receptor Mediates Larval Zebrafish Fin Duplication Following Exposure to Benzofluoranthenes.

The aryl hydrocarbon receptor (AHR) mediates developmental toxicity of several xenobiotic classes including polycyclic aromatic hydrocarbons. Using embryonic zebrafish, we previously identified 4 polycyclic aromatic hydrocarbons that caused a novel phenotype among AHR ligands-growth of a lateral, duplicate caudal fin fold. The window of sensitivity to the most potent inducer of this phenotype, benzo[k]fluoranthene (BkF), was prior to 36 h postfertilization (hpf), although the phenotype was not manifest until 60 hpf. AHR dependency via Ahr2 was demonstrated using morpholino knockdown. Hepatocyte ablation demonstrated that hepatic metabolism of BkF was not required for the phenotype, nor was it responsible for the window of sensitivity. RNA sequencing performed on caudal trunk tissue from BkF-exposed animals collected at 48, 60, 72, and 96 hpf showed upregulation of genes associated with AHR activation, appendage development, and tissue patterning. Genes encoding fibroblast growth factor and bone morphogenic protein ligands, along with retinaldehyde dehydrogenase, were prominently upregulated. Gene Ontology term analysis revealed that upregulated genes were enriched for mesoderm development and fin regeneration, whereas downregulated genes were enriched for Wnt signaling and neuronal development. MetaCore (Clarivate Analytics) systems analysis of orthologous human genes predicted that R-SMADs, AP-1, and LEF1 regulated the expression of an enriched number of gene targets across all time points. Our results demonstrate a novel aspect of AHR activity with implications for developmental processes conserved across vertebrate species.

Multidimensional chemobehavior analysis of flavonoids and neuroactive compounds in zebrafish.

The comparative analysis of complex behavioral phenotypes is valuable as a reductionist tool for both drug discovery and defining chemical bioactivity. Flavonoids are a diverse class of chemicals that elicit robust neuroactive and hormonal actions, though bioactivity information is limited for many, particularly for neurobehavioral endpoints. Here, we used a zebrafish larval chemomotor response (LCR) bioassay to comparatively evaluate a suite of 24 flavonoids, and in addition a panel of 30 model neuroactive compounds representing diverse modes of action (e.g. caffeine, chlorpyrifos, methamphetamine, nicotine, picrotoxin). Naïve larval zebrafish were exposed to concentration ranges of each compound at 120 hour post-fertilization (hpf) and locomotor activity measured for 5 h. The model neuroactive compounds were largely behaviorally bioactive (20 of 30) with most effects phenotypic of their known modes of action. Flavonoids rapidly and broadly elicited hyperactive locomotor effects (22 of 24). Multidimensional analyses compared responses over time and identified three distinct bioactive groups of flavonoids based on efficacy and potency. Using GABAergics to modulate hyperactive responses, two flavonoids, (S)-equol and kaempferol were tested for GABAA receptor antagonism, as well as a known GABAA receptor antagonist, picrotoxin. Pharmacological intervention with positive allosteric modulators of the GABAA receptor, alfaxalone and chlormethiazole, ameliorated the hyperactive response to picrotoxin, but not for (S)-equol or kaempferol. Taken together, these studies demonstrate that flavonoids are differentially bioactive and that the chemobehavioral effects likely do not involve a GABAA receptor mediated mode of action. Overall, the integrative zebrafish platform provides a useful framework for comparatively evaluating high-content chemobehavioral data for sets of structurally- and mechanistically-related flavonoids and neuroactive compounds.

AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish.

The aryl hydrocarbon receptor (AHR) is a conserved ligand-activated transcription factor required for proper vertebrate development and homeostasis. The inappropriate activation of AHR by ubiquitous pollutants can lead to adverse effects on wildlife and human health. The zebrafish is a powerful model system that provides a vertebrate data stream that anchors hypothesis at the genetic and cellular levels to observations at the morphological and behavioral level, in a high-throughput format. In order to investigate the endogenous functions of AHR, we generated an AHR2 (homolog of human AHR)-null zebrafish line (ahr2osu1) using the clustered, regulatory interspaced, short palindromic repeats (CRISPR)-Cas9 precision genome editing method. In zebrafish, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated toxicity requires AHR2. The AHR2-null line was resistant to TCDD-induced toxicity, indicating the line can be used to investigate the biological and toxicological functions of AHR2. The AHR2-null zebrafish exhibited decreased survival and fecundity compared to the wild type line. At 36 weeks, histological evaluations of the AHR2-null ovaries revealed a reduction of mature follicles when compared to wild type ovaries, suggesting AHR2 regulates follicle growth in zebrafish. AHR2-null adults had malformed cranial skeletal bones and severely damaged fins. Our data suggests AHR2 regulates some aspect(s) of neuromuscular and/or sensory system development, with impaired behavioral responses observed in larval and adult AHR2-null zebrafish. This study increases our understanding of the endogenous functions of AHR, which may help foster a better understanding of the target organs and molecular mechanisms involved in AHR-mediated toxicities.

Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System.

Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay-an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1-50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization.

Optimizing multi-dimensional high throughput screening using zebrafish.

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories.

PFOS, PFNA, and PFOA sub-lethal exposure to embryonic zebrafish have different toxicity profiles in terms of morphometrics, behavior and gene expression.

Polyfluorinated compounds (PFC) are a class of anthropogenic, persistent and toxic chemicals. PFCs are detected worldwide and consist of fluorinated carbon chains of varying length, terminal groups, and industrial uses. Previous zebrafish studies in the literature as well as our own studies have shown that exposure to these chemicals at a low range of concentrations (0.02-2.0µM; 20-2000ppb) resulted in chemical specific developmental defects and reduced post hatch survival. It was hypothesized that sub-lethal embryonic exposure to perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), or perfluorooctanoic acid (PFOA) would result in different responses with regard to morphometric, behavior, and gene expression in both yolk sac fry and larval zebrafish. Zebrafish were exposed to PFOS, PFOA, and PFNA (0.02, 0.2, 2.0µM) for the first five days post fertilization (dpf) and analyzed for morphometrics (5 dpf, 14 dpf), targeted gene expression (5 dpf, 14 dpf), and locomotive behavior (14 dpf). All three PFCs commonly resulted in a decrease in total body length, increased tfc3a (muscle development) expression and decreased ap1s (protein transport) expression at 5dpf, and hyperactive locomotor activity 14 dpf. All other endpoints measured at both life-stage time points varied between each of the PFCs. PFOS, PFNA, and PFOA exposure resulted in significantly altered responses in terms of morphometric, locomotion, and gene expression endpoints, which could be manifested in field exposed teleosts.

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium.

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes.

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose-response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17ß-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER ßa, ER ßb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17ß-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10ng/g 17ß-estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17ß-estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen-responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.

Zebrafish: A marvel of high-throughput biology for 21st century toxicology.

The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing.

Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects.

Retinoic acid (RA) is involved in multifarious and complex functions necessary for vertebrate development. RA signaling is reliant on strict enzymatic regulation of RA synthesis and metabolism. Improper spatiotemporal expression of RA during development can result in vertebrate axis defects. microRNAs (miRNAs) are also pivotal in orchestrating developmental processes. While mechanistic links between miRNAs and axial development are established, the role of miRNAs in regulating metabolic enzymes responsible for RA abundance during axis formation has yet to be elucidated. Our results uncovered a role of miR-19 family members in controlling RA metabolism through the regulation of CYP26A1 during vertebrate axis formation. Global miRNA expression profiling showed that developmental RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. A reporter assay confirmed that cyp26a1 is a bona fide target of miR-19 in vivo. Transient knockdown of miR-19 phenocopied axis defects caused by RA exposure. Exogenous miR-19 rescued the axis defects induced by RA exposure. Taken together, these results indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 expression and subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development via regulation of RA signaling.

Inhibition of vitellogenin gene induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin is mediated by aryl hydrocarbon receptor 2 (AHR2) in zebrafish (Danio rerio).

Vitellogenins are hepatically derived yolk-protein precursors required for oogenesis in all oviparous teleosts. Altered gene-regulation of vitellogenesis by environmental contaminants can have profound effects on reproductive success, and ultimately population sustainability. To better understand chemical effects on vitellogenin gene regulation, we tested the hypothesis that activation of the aryl hydrocarbon receptor 2 (AHR2) by dioxin inhibits the estrogen receptor pathway regulation of 3 vitellogenin genes (vtg1-3) in vivo, using zebrafish (Danio rerio) as a model teleost. Using an embryo-larval bioassay, embryos were either treated with 1000 pptr (parts-per-trillion, pg/mL) 17α-ethynylestradiol (EE2) alone from 6h post fertilization (hpf) to 4 days post fertilization (dpf), or pre-treated with dioxin (4-5 hpf) prior to EE2. Pre-treatment with 400 pptr 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) or 1,2,3,7,8-pentachlorodibenzo-p-dioxin inhibited the EE2 induction of vtg1, vtg2 and vtg3 by >95% (p≤0.05). In comparison, a splice-blocking AHR2 morpholino used to down-regulate ahr2 expression significantly reduced the inhibition of vtg1, vtg2 and vtg3 by 400 pptr 2,3,7,8-TCDD (20.7-27.4% rescue). These studies demonstrate that 2,3,7,8-TCDD directly inhibits the vitellogenin pathway in vivo through activation of the AHR2. This work provides evidence for AHR2 dependent cross-talk inhibition of vitellogenin genes and offers insight into anti-estrogenic reproductive effects observed in oviparous species exposed to AHR agonist contaminants.

Decreased vitellogenin inducibility and 17ß-estradiol levels correlated with reduced egg production in killifish (Fundulus heteroclitus) from Newark Bay, NJ.

Aquatic species inhabiting polluted estuaries are exposed to complex mixtures of xenobiotics which can alter normal reproduction. We previously reported that female Atlantic killifish (Fundulus heteroclitus) from the highly contaminated Newark Bay, NJ (USA) exhibited an inhibition of oocyte development due to reduced vitellogenin (egg-yolk precursor) levels. Our hypothesis was that the inhibition of oocyte development in Newark Bay killifish is due to (1) deficient levels of circulating 17ß-estradiol, and (2) a decreased sensitivity of the vitellogenin pathway to physiological doses of 17ß-estradiol. In the first study, adult naïve killifish from Tuckerton, NJ (reference) were caged at Tuckerton and Newark Bay. After 1 month, males caged at Newark Bay exhibited inductions of hepatic vitellogenin and estrogen receptor α, which were transient and returned to basal levels after 2 months (p≤0.05). In the second study, fecundity and 17ß-estradiol levels were measured in reproductively active adult females from Tuckerton and Newark Bay. Tuckerton females produced 140 eggs per female and Newark Bay females produced 11 eggs per female. Embryos from Newark Bay had 34% greater mortality and 28% less hatch, relative to Tuckerton. In addition, embryo mass and yolk-volume of Newark Bay embryos compared to Tuckerton embryos was 16% and 25% lower, respectively. Circulating 17ß-estradiol levels in Newark Bay females (0.26 ng/mL) were measured to be 8-fold lower than Tuckerton females (2.25 ng/mL). In the third study, adult killifish from both sites were dosed with 17ß-estradiol to assess the sensitivity of the vitellogenin pathway. At doses of 0.01, 0.1, 1 and 10 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay males were significantly inhibited by 97, 99, 98 and 44%, respectively, compared to Tuckerton males. At doses of 0.01, 0.1, 1, 10 and 100 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay females were inhibited by 89, 79, 61, 40 and 30%, respectively, compared to Tuckerton females. These differences in inducibility could not be explained by altered hepatic expression of estrogen receptors α, ßa or ßb. Based on the caged and dose-response studies, contaminants that down-regulate vitellogenin would interfere with its ability to be used as a biomarker for xeno-estrogen exposures. These studies demonstrate that contaminants within Newark Bay exert both estrogenic and anti-estrogenic responses which results in an overtly anti-estrogenic phenotype (reduced egg production due to inhibition of vitellogenesis).

Impaired reproductive health of killifish (Fundulus heteroclitus) inhabiting Newark Bay, NJ, a chronically contaminated estuary.

A battery of biomarkers were used to evaluate the reproductive health and contaminant exposure of Atlantic killifish (Fundulus heteroclitus) inhabiting the heavily industrialized Newark Bay and a reference population from Great Bay, Tuckerton, NJ. The biomarkers investigated included classical endpoints (gonad and liver histopathology, body and tissue morphometrics), hepatic mRNA expression (CYP1A and vitellogenin I), hepatic protein levels (CYP1A and vitellogenin), gonadal aromatase mRNA expression, and chemical exposure analyses (bile PAHs). Our data showed no significant differences between populations for body size and body weight. However, Newark Bay killifish exhibited molecular and morphological changes indicative of impaired reproductive health and endocrine disruption compared to the reference population. Newark Bay males had decreased gonad weight, altered testis development and decreased gonadal aromatase mRNA expression. Newark Bay females had decreased gonad weight, inhibited gonadal development, decreased hepatic vitellogenin production (mRNA and protein) and increased mRNA expression of gonadal aromatase. In addition, Newark Bay females had a significant increase in the percent of pre-vitellogenic follicles (43% at Tuckerton, 64% at Newark Bay) and a significantly decreased percent of follicles at the mid-vitellogenic and mature stages (25% mature at Tuckerton and 3% at Newark Bay). In addition to reproductive endpoints, killifish at Newark Bay exhibited high basal levels of CYP1A mRNA and protein expression which indicated exposure to aryl hydrocarbon receptor (AhR) agonists. An inverse relationship between hepatic CYP1A protein and hepatic vitellogenin mRNA expression was established suggesting a possible link between AhR agonist exposure and vitellogenesis. Killifish in the NY-NJ Harbor Estuary are exposed to a number of chemicals that can interact with the AhR pathway and stimulate enzymatic activity along with chemicals that can modify reproductive success in this indigenous species. Similar effects on the reproductive development in less resilient species may limit their ability to repopulate the NY-NJ Harbor Estuary and similarly contaminated water systems.