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Thyroid hormones (TH) are essential for normal brain development, influencing neural cell differentiation, migration, and synaptogenesis. Multiple endocrine-disrupting chemicals (EDCs) are found in the environment, raising concern for their potential effects on TH signaling and the consequences on neurodevelopment and behavior. While most research on EDCs investigates the effects of individual chemicals, human health may be adversely affected by a mixture of chemicals. The potential consequences of EDC exposure on human health are far-reaching and include problems with immune function, reproductive health, and neurological development. We hypothesized that embryonic exposure to a mixture of chemicals (containing phenols, phthalates, pesticides, heavy metals, and perfluorinated, polychlorinated, and polybrominated compounds) identified as commonly found in the human amniotic fluid could lead to altered brain development. We assessed its effect on TH signaling and neurodevelopment in an amphibian model (Xenopus laevis) highly sensitive to thyroid disruption. Fertilized eggs were exposed for eight days to either TH (thyroxine, T4 10 nM) or the amniotic mixture (at the actual concentration) until reaching stage NF47, where we analyzed gene expression in the brains of exposed tadpoles using both RT-qPCR and RNA sequencing. The results indicate that whilst some overlap on TH-dependent genes exists, T4 and the mixture have different gene signatures. Immunohistochemistry showed increased proliferation in the brains of T4-treated animals, whereas no difference was observed for the amniotic mixture. Further, we demonstrated diminished tadpoles’ motility in response to T4 and mixture exposure. As the individual chemicals composing the mixture are considered safe, these results highlight the importance of examining the effects of mixtures to improve risk assessment.

Thyroid hormones (THs) are essential for normal brain development, influencing neural cell differentiation, migration, and synaptogenesis. Multiple endocrine-disrupting chemicals (EDCs) are found in the environment, raising concern for their potential effects on thyroid hormone signaling and the consequences on neurodevelopment and behavior. While most research on EDCs investigates the effects of individual chemicals, human health may be adversely affected by a mixture of chemicals. Many compounds belonging to a wide range of chemical classes have been identified as EDCs, notably those affecting thyroid hormone signaling. We hypothesized that embryonic exposure to a mixture of chemicals (containing phenols, phthalates, pesticides, heavy metals, perfluorinated -, polychlorinated, and polybrominated compound) commonly found in the human amniotic fluid could lead to altered brain development to assess its effect on thyroid hormone signaling and neurodevelopment in an amphibian model (Xenopus laevis), highly sensitive to thyroid disruption. Newly hatched tadpoles were exposed for eight days to either TH (thyroxine, T4 10nM) or the amniotic mixture (1x concentration) and gene expression was analyzed in the brains of exposed tadpoles using both RT-qPCR and RNA sequencing. Results indicate that whilst some overlap on TH-dependent genes exist, T4 and the mixture have different gene signatures. Immunohistochemistry showed increased proliferation in the brains of T4-treated animals whereas no difference was observed for the amniotic mixture. Further, we demonstrated diminished tadpoles’ motility in response to T4 and mixture exposure. As the individual chemicals composing the mixture are considered safe, these results highlight the importance of examining the effects of mixtures to improve risk assessment

International audience; North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4′–OH–PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4′–OH–PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4′–OH–PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4′–OH–PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4′–OH–PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4′–OH–PPF alone. We conclude that 4′–OH–PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

Significance Many environmental pollutants act as endocrine disruptors that interfere with normal endocrine regulation and promote adverse effects in humans. As a major target of xenobiotics, the pregnane X receptor (PXR) is known to play opposite roles by both facilitating their clearance and mediating their toxic effects. Here, we use structural and functional approaches to describe two converging mechanisms leading to a robust synergistic stimulation of the PXR pathway by mixtures of three chemicals exhibiting very low efficacy when administered separately. This “cocktail effect” relies on two cooperative binding processes that enhance both ligand binding affinity and recruitment of transcriptional coactivators. Our findings show how chemical mixtures may alter physiology and homeostasis at concentrations where individual components are considered safe., Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR–PXR and potentially several other nuclear receptor heterodimers.

Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 μm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation.

International audience; Accessibility and imaging of cell compartments in big specimens are crucial for cellular biological research but also a matter of contention. Confocal imaging and tissue clearing on whole organs allow for 3D imaging of cellular structures after being subjected to in-toto immunohistochemistry. Lately, the passive CLARITY technique (PACT) has been adapted to clear and immunolabel large specimens or individual organs of several aquatic species. We recently demonstrated tissue clearing on one-week old tadpole brain (Fini et al., Sci Rep 7:43786, 2017). We here describe a further simplified version with clearing of small tissue samples (thickness inferior to 500 μm)) carried out by immersion in a fructose-based high-refractive index solution (fbHRI). By refining steps of the protocol, we were able to reduce the overall procedure time by two thirds. This offers the advantages of reducing the time of experimentation to a week and minimizes procedure-induced tissue deformations. This protocol can be easily adapted to be performed on thick section. We present an example of immunohistochemistry performed on NF45 Xenopus laevis brains with anti-pH 3 (phosphorylated histone H3) antibody used to stain chromatin condensation commonly associated with proliferation.

Xenopus is an excellent model for studying thyroid hormone signaling as it undergoes thyroid hormone–dependent metamorphosis. Despite the fact that receptors and deiodinases have been described in Xenopus, membrane transporters for these hormones are yet to be characterized. We cloned Xenopus monocarboxylate transporter 8 (mct8) and organic anion-transporting polypeptide 1C1 (oatpc1c1), focusing on these two transporters given their importance for vertebrate brain development. Protein alignment and bootstrap analysis showed that Xenopus mct8 and oatp1c1 are closer to their mammalian orthologs than their teleost counterparts. We functionally characterized the two transporters using a radiolabeled hormones in vitro uptake assay in COS-1 cells. Xenopus mct8 was found to actively transport both T3 and T4 bidirectionally. As to the thyroid precursor molecules, diiodotyrosine (DIT) and monoiodotyrosine (MIT), both human and Xenopus mct8, showed active efflux, but no influx. Again similar to humans, Xenopus oatp1c1 transported T4 but not T3, MIT, or DIT. We used reverse transcription quantitative polymerase chain reaction and in situ hybridization to characterize the temporal and spatial expression of mct8 and oatp1c1 in Xenopus. Specific expression of the transporter was observed in the brain, with increasingly strong expression as development progressed. In conclusion, these results show that Xenopus thyroid hormone transporters are functional and display marked spatiotemporal expression patterns. These features make them interesting targets to elucidate their roles in determining thyroid hormone availability during embryonic development.

While numerous detection methods exist for environmental heavy metal monitoring, easy-to-use technologies combining rapidity with in vivo measurements are lacking. Multiwell systems exploiting transgenic tadpoles are ideal but require time-consuming placement of individuals in wells. We developed a real-time flow-through system, based on Fountain Flow cytometry, which measures in situ contaminant-induced fluorescence in transgenic amphibian larvae immersed in water samples. The system maintains the advantages of transgenic amphibians, but requires minimal human intervention. Portable and self-contained, it allows on-site measurements. Optimization exploited a transgenic Xenopus laevis bearing a chimeric gene with metal responsive elements fused to eGFP. The transgene was selectively induced by 1 microM Zn(2+). Using this tadpole we show the continuous flow method to be as rapid and sensitive as image analysis. Flow-through readings thus accelerate the overall process of data acquisition and render fluorescent monitoring of tadpoles suitable for on-site tracking of heavy metal pollution.

Thyroid hormone receptors generally activate transcription of target genes in the presence of thyroid hormone (T(3)) and repress their transcription in its absence. Here, we investigated the role of unliganded thyroid hormone receptor (TR) during vertebrate development using an amphibian model. Previous studies led to the hypothesis that before production of endogenous T(3), the presence of unliganded receptor is essential for premetamorphic tadpole growth. To test this hypothesis, we generated a Xenopus laevis TR beta mutant construct ineffective for gene repression owing to impaired corepressor NCoR recruitment. Overexpression by germinal transgenesis of the mutant receptor leads to lethality during early development with numerous defects in cranio-facial and eye development. These effects correlate with TR expression profiles at these early stages. Molecular analysis of transgenic mutants reveals perturbed expression of genes involved in eye development. Finally, treatment with iopanoic acid or NH-3, modulators of thyroid hormone action, leads to abnormal eye development. In conclusion, the data reveal a role of unliganded TR in eye development.

A key event in metamorphosis of anuran amphibians is tail resorption. This composite structure includes epidermal cells, spinal cord, muscle fibres and connective tissue. It is unclear how resorption proceeds and to what extent the signals for the death process are transmitted between cells. We determined the kinetics of metamorphosis, apoptosis, and tail regression in the diploid anuran, Xenopus tropicalis, a species more suited to genetic analysis than the pseudotetraploid, Xenopus laevis. Metamorphosis was found to proceed at a regular and predictable rate in X. tropicalis but not in X. laevis. Caspase 3 activity and mRNA levels were correlated with TdT-mediated dUTP nick end-labeling (TUNEL) signalling and most markedly increased in tail muscle and spinal cord. It has been proposed that muscles die as a result of loss of connectivity with the surrounding matrix. To test this hypothesis, we used direct DNA injection in trunk and tail muscle to overexpress Xenopus Bcl-XL (xR11), an anti-apoptotic gene, along with a marker gene (luciferase or GFP). xR11 significantly inhibited the cell death process in both trunk and tail muscle. This protection was functional even up to stage 64 on completion of tail regression. We conclude that (1) somatic gene transfer can be applied to analyse cell fate in X. tropicalis, and (2) that muscle death can be abrogated despite extracellular matrix loss. © 2002 Wiley-Liss, Inc.

International audience; The flame retardant tetrabromobisphenol A (TBBPA) is a high production flame retardant that interferes with thyroid hormone (TH) signaling. Despite its rapid metabolism in mammals, TBBPA is found in significant amounts in different tissues. Such findings highlight first a need to better understand the effects of TBBPA and its metabolites and second the need to develop models to address these questions experimentally. We used Xenopus laevis tadpoles to follow radiolabeled (14)C-TBBPA uptake and metabolism. Extensive and rapid uptake of radioactivity was observed, tadpoles metabolizing > 94% of (14)C-TBBPA within 8 h. Four metabolites were identified in water and tadpole extracts: TBBPA-glucuronide, TBBPA-glucuronide-sulfate, TBBPA-sulfate, and TBBPA-disulfate. These metabolites are identical to the TBBPA conjugates characterized in mammals, including humans. Most radioactivity (> 75%) was associated with sulfated conjugates. The antithyroid effects of TBBPA and the metabolites were compared using two in vivo measures: tadpole morphology and an in vivo tadpole TH reporter gene assay. Only TBBPA, and not the sulfated metabolites, disrupted thyroid signaling. Moreover, TBBPA treatment did not affect expression of phase II enzymes involved in TH metabolism, suggesting that the antithyroid effects of TBBPA are not due to indirect effects on TH metabolism. Finally, we show that only the parent TBBPA inhibits T3-induced transactivation in cells expressing human, zebrafish, or X. laevis TH receptor, TRα. We conclude, first, that perturbation of thyroid signaling by TBBPA is likely due to rapid direct action of the parent compound, and second, that Xenopus is an excellent vertebrate model for biotransformation studies, displaying homologous pathways to mammals.

The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.

Increasing numbers of substances present in the environment are postulated to have endocrine-disrupting effects on vertebrate populations. However, data on disruption of thyroid signaling are fragmentary, particularly at the molecular level. Thyroid hormone (TH; triiodothyronine, T3) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, T3 orchestrates amphibian metamorphosis, thereby providing an exceptional model for identifying thyroid-disrupting chemicals. We combined these two advantages to develop a method for following and quantifying the transcriptional action of T3 in Xenopus laevis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to "on-line" imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with TH-responsive elements coupled to either luciferase or green fluorescent protein to follow T3-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pretreatment protocol that induced competence to respond to T3 and thus to assess T3 effects and T3 disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak (10(-12) M) pulse of T3 that induced TH receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid-disrupting activity of environmentally relevant concentrations (10(-8) M) of acetochlor, a persistent herbicide.

International audience; Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Apoptosis is fundamental to normal vertebrate development. A dramatic example of postembryonic development involving apoptosis is tail regression during amphibian metamorphosis. Earlier studies led us to propose a functional role for the pro-apoptotic protein Bax in tadpole tail regression. However, its physiological relevance has never been analyzed. We have now cloned a cDNA encoding Xenopus laevis bax (xlbax) and used in vivo gene transfer in tail muscle to analyze the effects of xlbax overexpression. Furthermore, by using an antisense strategy in a similar experimental paradigm, xlbax antisense mRNA was shown to block the apoptotic effects of xlbax and protect against apoptosis in metamorphosing tadpoles. Our results suggest that xlbax is a regulator of muscle fiber death in the regressing tail during metamorphosis.

A key event in metamorphosis of anuran amphibians is tail resorption. This composite structure includes epidermal cells, spinal cord, muscle fibres and connective tissue. It is unclear how resorption proceeds and to what extent the signals for the death process are transmitted between cells. We determined the kinetics of metamorphosis, apoptosis, and tail regression in the diploid anuran, Xenopus tropicalis, a species more suited to genetic analysis than the pseudotetraploid, Xenopus laevis. Metamorphosis was found to proceed at a regular and predictable rate in X. tropicalis but not in X. laevis. Caspase 3 activity and mRNA levels were correlated with TdT-mediated dUTP nick end-labeling (TUNEL) signalling and most markedly increased in tail muscle and spinal cord. It has been proposed that muscles die as a result of loss of connectivity with the surrounding matrix. To test this hypothesis, we used direct DNA injection in trunk and tail muscle to overexpress Xenopus Bcl-X(L) (xR11), an anti-apoptotic gene, along with a marker gene (luciferase or GFP). xR11 significantly inhibited the cell death process in both trunk and tail muscle. This protection was functional even up to stage 64 on completion of tail regression. We conclude that (1) somatic gene transfer can be applied to analyse cell fate in X. tropicalis, and (2) that muscle death can be abrogated despite extracellular matrix loss.

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X L plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-X L homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

International audience; Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.