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Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio).

ABSTRACT: Thyroid hormones (THs) regulate neurodevelopment, thus TH disruption is widely posited as a mechanism of developmental neurotoxicity for diverse environmental chemicals. Zebrafish have been proposed as an alternative model for studying the role of TH in developmental neurotoxicity. To realize this goal, it is critical to characterize the normal ontogenetic expression profile of TH signaling molecules in the developing zebrafish and determine the sensitivity of these molecules to perturbations in TH levels. To address these gaps in the existing database, we characterized the transcriptional profiles of TH transporters, deiodinases (DIOs), receptors (TRs), nuclear coactivators (NCOAs), nuclear corepressors (NCORs), and retinoid X receptors (RXRs) in parallel with measurements of endogenous TH concentrations and tsh? mRNA expression throughout the first five days of zebrafish development. Transcripts encoding these TH signaling components were identified and observed to be upregulated around 48-72?h post fertilization (hpf) concurrent with the onset of larval production of T4. Exposure to exogenous T4 and T3 upregulated mct8, dio3-b, tr?-a, tr?, and mbp-a levels, and downregulated expression of oatp1c1. Morpholino knockdown of TH transporter mct8 and treatment with 6-propyl-2-thiouracil (PTU) was used to reduce cellular uptake and production of TH, an effect that was associated with downregulation of dio3-b at 120?hpf. Collectively, these data confirm that larval zebrafish express orthologs of TH signaling molecules important in mammalian development and suggest that there may be species differences with respect to impacts of TH disruption on gene transcription.

SUBMITTER: Walter KM

PROVIDER: S-EPMC6331280 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio).

Walter Kyla M KM Miller Galen W GW Chen Xiaopeng X Yaghoobi Bianca B Puschner Birgit B Lein Pamela J PJ

General and comparative endocrinology 20181115

Thyroid hormones (THs) regulate neurodevelopment, thus TH disruption is widely posited as a mechanism of developmental neurotoxicity for diverse environmental chemicals. Zebrafish have been proposed as an alternative model for studying the role of TH in developmental neurotoxicity. To realize this goal, it is critical to characterize the normal ontogenetic expression profile of TH signaling molecules in the developing zebrafish and determine the sensitivity of these molecules to perturbations in ...[more]

PMID: 30448381

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Project description:Thyroid hormones (THs) are essential for normal vertebrate development and diverse environmental chemicals are hypothesized to cause developmental toxicity by disrupting TH-mediated signaling. The larval zebrafish (Danio rerio) is an emerging in vivo model of developmental TH disruption; however, the effects of TR antagonism have not yet been studied in zebrafish. NH3, generally considered a potent and specific thyroid hormone receptor (TR) antagonist, has been used in rodents and Xenopus laevis to characterize phenotypes of TR antagonism. The objective of this study is to determine the effects of NH3 on endpoints previously determined to be TH-sensitive in larval zebrafish, specifically teratology and mortality, photomotor behavior, and mRNA expression of TH signaling genes. Zebrafish embryos were exposed to NH3 via static waterborne exposure at concentrations ranging from 0.001 to 10 μM beginning at 6 h post-fertilization (hpf) through 5 days post fertilization (dpf). Significant mortality and teratogenesis was observed at 3, 4, and 5 dpf in zebrafish exposed to NH3 at 10 μM. At concentrations that did not cause significant mortality, NH3 did not exert a consistent antagonistic effect on photomotor behavior assays or mRNA expression when administered alone or in the presence of exogenous T4. Rather, depending on the NH3 concentration and larval age NH3 decreased or increased swimming triggered by transition from light to dark. Similarly, inconsistent antagonistic and agonistic effects on mRNA expression of TH signaling genes were noted following treatment with NH3 alone. NH3 did inhibit T4 (30 nM)-induced gene expression; however, this was only consistently observed at a concentration of NH3 (10 μM) that also caused significant mortality. Collectively, these results suggest that NH3 does not act solely as a TR antagonist in larval zebrafish, but instead exhibits complex modulatory effects on TR activity. These data support the hypothesis that NH3 is a selective thyroid hormone receptor modulator. Further studies of NH3 interactions with the zebrafish thyroid hormone receptor are required to characterize the activity of NH3 in target tissues of the larval zebrafish at the molecular level, highlighting the importance of characterizing NH3 effects in specific models of TH-disruption to better interpret its actions in mechanistic screens of environmental chemicals for TH action.

Project description:Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 μM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 μM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 μM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 μM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 μM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.

Dataset Information

Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio).

Publications

Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio).

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