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