Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos.

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

Thyroid hormone action and disruption in the brain: Xenopus as a model to study disruption of thyroid hormone availability on early brain development

Thyroid hormones (TH) are essential for normal brain development in vertebrates. Disruption of TH action, either due to genetic and/or environmental factors, has been implicated in neurological defects such as autism, attention deficit hyperactivity disorders (ADHD) and IQ loss. On average, over 30 anthropogenic chemicals are present in all American women, with 15 being ubiquitous, including in pregnant women. Many of these chemicals are demonstrated or suspected TH disruptors. A representative mixture of these 15 common chemicals was established at concentrations reported in human amniotic fluid and an in vivo larval reporter (GFP) assay was used to determine integrated TH transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. Gene expression analysis on dissected brains showed significant changes in TH related genes including receptors, deiodinases and neural differentiation markers. Immunohistochemistry confirmed the mixture exposure action on neural proliferation as well as neuron and oligodendrocyte size. Mixture exposed tadpoles also displayed dose-dependent reductions in mobility. As TH signalling is strongly conserved across vertebrates these results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development. Genetic disruption is also evident in the Allan-Herndon-Dudley (AHD) syndrome where, in humans, the mutation of the brain specific TH transporter (THT), monocarboxylate transporter 8 (MCT8), leads to severe intellectual disability. The lack of neurological phenotype in mice model of Mct8 deficiency has driven the need for characterisation and generation of various non-rodent models. The Xenopus mct8 and additional TH transporters of interest were cloned and characterised. Protein alignment and bootstrap analysis showed that Xenopus mct8 is closer to its mammalian orthologue than its teleost counterparts. Radiolabelled hormones in vitro uptake assay confirmed Xenopus mct8 active transport of both T3 and T4 bi-directionally. As to the thyroid precursor molecules, diiodotyrosine (DIT) and monoiodotyrosine (MIT), both human and Xenopus mct8, showed active efflux, but no influx. Using real-time quantitative PCR (RT-qPCR) and in situ hybridization, specific expression of the mct8 was observed in the brain, with increasingly strong expression as development progressed. Using the before mentioned in vitro assay, effects of common environmental xenobiotics on MCT8 function were assessed. In conclusion, these results show that Xenopus TH transporters are functional and display marked spatio temporal expression patterns. These features make them interesting targets to elucidate their roles in determining TH availability during embryonic development. Xenopus laevis was used a model of choice in these experiments. X. laevis is ideal for studying TH signalling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. Further, X. laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Reference genes are essential for gene expression analysis when using RT-qPCR, and there is a lack of ad hoc reference genes for X. laevis. Using previously published RNA-seq data on different X. laevis stages, top 14 candidate gene were identified with respect to their expression levels as a function of developmental stage and degree of variation. Using RT-qPCR, the stability of these genes was evaluated and optimized reference gene pair combinations established for studying 1) development (early whole embryos), 2) brains at later stages (metamorphosis and adult), and 3) thyroid signalling

Thyroid-disrupting chemicals and brain development: an update.

This review covers recent findings on the main categories of thyroid hormone-disrupting chemicals and their effects on brain development. We draw mostly on epidemiological and experimental data published in the last decade. For each chemical class considered, we deal with not only the thyroid hormone-disrupting effects but also briefly mention the main mechanisms by which the same chemicals could modify estrogen and/or androgen signalling, thereby exacerbating adverse effects on endocrine-dependent developmental programmes. Further, we emphasize recent data showing how maternal thyroid hormone signalling during early pregnancy affects not only offspring IQ, but also neurodevelopmental disease risk. These recent findings add to established knowledge on the crucial importance of iodine and thyroid hormone for optimal brain development. We propose that prenatal exposure to mixtures of thyroid hormone-disrupting chemicals provides a plausible biological mechanism contributing to current increases in the incidence of neurodevelopmental disease and IQ loss

Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis.

Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis

Functional Characterization of Xenopus Thyroid Hormone Transporters mct8 and oatp1c1.

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