Search

Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10−12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.

The monocarboxylate transporter 8 (MCT8) is a specific thyroid hormone transporter and plays an essential role in fetal development. Inactivating mutations in the MCT8 encoding gene SLC16A2 (solute carrier family 16, member 2) lead to the Allan-Herndon-Dudley syndrome, a condition presenting with severe endocrinological and neurological phenotypes. However, the cellular distribution pattern and dynamic expression profile are still not well known for early human neural development. Objective Development and characterization of fluorescent MCT8 reporters that would permit live-cell monitoring of MCT8 protein expression in vitro in human induced pluripotent stem cell (hiPSC)-derived cell culture models. Methods A tetracysteine (TC) motif was introduced into the human MCT8 sequence at four different positions as binding sites for fluorescent biarsenical dyes. Human Embryonic Kidney 293 cells were transfected and stained with fluorescein-arsenical hairpin-binder (FlAsH). Counterstaining with specific MCT8 antibody was performed. Triiodothyronine (T3) uptake was indirectly measured with a T3 responsive luciferase-based reporter gene assay in Madin-Darby Canine Kidney 1 cells for functional characterization. Results FlAsH staining and antibody counterstaining of all four constructs showed cell membrane expression of all MCT8 constructs. The construct with the tag after the first start codon demonstrated comparable T3 uptake to the MCT8 wildtype. Conclusion Our data indicate that introduction of a TC-tag directly after the first start codon generates a MCT8 reporter with suitable characteristics for live-cell monitoring of MCT8 expression. One promising future application will be generation of stable hiPSC MCT8 reporter lines to characterize MCT8 expression patterns during in vitro neuronal development.

Whole exome sequencing discovers causative mutations in less than 50 % of rare disease patients, suggesting the presence of additional mutations in the non-coding genome. So far, non-coding mutations have been identified in less than 0.2 % of individuals with genetic diseases listed in the ClinVar database and exhibit highly diverse molecular mechanisms. In contrast to our capability to sequence the whole genome, our ability to discover and functionally confirm such non-coding mutations is lagging behind severely. We discuss the problems and present examples of confirmed mutations in deep intronic sequences, non-coding triplet repeats, enhancers, and larger structural variants and highlight their proposed disease mechanisms. Finally, we discuss the type of data that would be required to establish non-coding mutation detection in routine diagnostics.

Background: Defects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns, but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification, and subsequent morphogenetic processes in thyroid development. Methods: To begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small-molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. Results: Systematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding, as well as hypoplastic, enlarged, or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified bone morphogenetic protein and fibroblast growth factor signaling as key factors for thyroid specification and early thyroid organogenesis, highlighted the importance of low Wnt activities during early development for thyroid specification, and implicated drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. Conclusions: By integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken, and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases., info:eu-repo/semantics/published

Context Pro-opiomelanocortin (POMC) and the melanocortin-4 receptor (MC4R) play a pivotal role in the leptin–melanocortin pathway. Mutations in these genes lead to monogenic types of obesity due to severe hyperphagia. In addition to dietary-induced obesity, a cardiac phenotype without hypertrophy has been identified in MC4R knockout mice. Objective We aimed to characterize cardiac morphology and function as well as tissue Na+ content in humans with mutations in POMC and MC4R genes. Methods A cohort of 42 patients (5 patients with bi-allelic POMC mutations, 6 heterozygous MC4R mutation carriers, 19 obese controls without known monogenic cause, and 12 normal weight controls) underwent cardiac magnetic resonance (CMR) imaging and 23Na-MRI. Results Monogenic obese patients with POMC or MC4R mutation respectively had a significantly lower left ventricular mass/body surface area (BSA) than nonmonogenic obese patients. Left ventricular end-diastolic volume/BSA was significantly lower in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. Subcutaneous fat and skin Na+ content was significantly higher in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. In these compartments, the water content was significantly higher in patients with POMC and MC4R mutation than in control groups. Conclusion Patients with POMC or MC4R mutations carriers had a lack of transition to hypertrophy, significantly lower cardiac muscle mass/BSA, and stored more Na+ within the subcutaneous fat tissue than nonmonogenic obese patients. The results point towards the role of the melanocortin pathway for cardiac function and tissue Na+ storage and the importance of including cardiologic assessments into the diagnostic work-up of these patients.

1. Introduction Assessing risk factors from data needs statistical modeling of that data. Here, mostly generalized linear models are applied, including logistic regression, and Poisson regression. The used framework struggles when the data set at hand is not a low-dimensional problem (i.e. only consists of only a few features), the data generating model is unknown and should a bit more ambitious than just a very simple linear one. Very often, we have data sets with almost as many, or even more variables than data points, and categorical features with many classes increase this problem for linear models even more (i.e. by inflating the design matrix with dummy varaibles). The classical framework fails here completely, as it has very high requirements on the data in order to successfully do a data analysis. And if the data generation model is unknown and we have to somehow guess a good model, it only gets worse. Here, we want to investigate the vast possibilities of Machine Learning (also known as statistical Learning, predictive modeling, and algorithmic modeling). On the field of machine learning, we often encounter data sets that are difficult to handle when using the classical statistical framework. There is the preconception, Machine Learning (ML) only produces predictive, but non-interpretable models, so-called black boxes. However, this was only ever the case for certain model types. Machine learning has produced highly interpretable models, such as the decision trees. But there aremany other ML models, which innately deliver valuable information about the data at hand. Moreover, this way of thinking is somewhat narrow in the classical framework, since it only wants to deal with a simple interpretation of the fitted values of its simplistic model. When we consider the kind of very complex models we are dealing with in physics, for example - Einstein's field equation, quantum field theories, but also the modeling of whole proteins and biochemical pathways - the way models are interpreted and the focus on oversimplified linear models must be reconsidered. The focus of the ML models is on their predictive capabilities. This is similar to many other sciences, but different to the classical statistical modelling. However, in physics we want to create a model from first principles, our focus in the application of machine laerning models is on their predictivity. The cat has to catch mice, no matter what color the cat is. If it is successfull, we would also accept a dog for the job. This might sound odd or just wrong for the classical statistical modelling tradition. Nevertheless, we want to emphasize that both ways, ML and the classical way, just lead to empirical ad-hoc models, as physicist would see them. What we do here is more interpolation than modelling. This is not intrinsically bad, but it means that the quest for the “right” model will lead to nowhere. We just need a good model. If “good” means good predictivity, than we should try the ML framework. But, we also need a model that gives us an insight into the world that is represented by the data.

Significance Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. These motifs belong to the most abundant recognition motifs in the eukaryotic genome and preferentially adopt a left-handed polyproline helix II, a secondary structure element that has been notoriously difficult to mimic with small molecules. Here, we present a structure-guided design effort yielding a toolkit of chemical entities that enables rational construction of selective small molecule inhibitors for these protein domains. We succeeded in developing an inhibitor for the Ena/VASP protein family that is active in vivo and reduces extravasation of invasive breast cancer cells in a zebrafish model., Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

Summary The thyroid plays an essential role in homeostasis and development, but the extrinsic regulation of its embryonic development remains poorly understood. Recently, we have identified the FGF and BMP pathways to be crucial for thyroid specification and have confirmed the hypothesis that the cardiac mesoderm provides the FGF and BMP ligands to regulate this process. However, it is not clear how these ligands control thyroid specification. To study the molecular mechanisms underlying early thyroid development, we combined a pharmacological approach in zebrafish embryos with genetic models, to modulate the activity of the FGF and BMP pathways at different embryonic stages. We first characterized the expression of the transcription factors pax2a and nkx2.4b - the two main early thyroid markers - in the anterior foregut endoderm and observed that pax2a was expressed from 18 hours post fertilization (hpf) and marked a large endodermal cell population while nkx2.4b was expressed from 24 hpf and marked only a subset of the pax2a-positive endodermal cells. Interestingly, the activity profiles of FGF and BMP coincided with the detection of pax2a and nkx2.4b expression, respectively. Brief modulations of the FGF and/or BMP pathways support the hypothesis that the FGF pathway regulates the expression of pax2a and the BMP pathway regulates the expression of nkx2.4b. Furthermore, inhibition of the BMP pathway during early segmentation has dramatic effects on thyroid specification, probably via the FGF pathway. Together with our previous observations, we propose here, an updated model of early thyroid development in which the foregut endoderm receives several synchronized waves of FGF and BMP signals from the cardiac mesoderm, which result in sequential activation of pax2a and nkx2.4b gene expression and subsequent thyroid specification.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

BackgroundCongenital hypothyroidism (CH) due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the far majority of cases. This situation reflects in part our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification.MethodsWe treated zebrafish embryos during different developmental periods with small molecule compounds known to modulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm and cardiovascular development using whole mountin situhybridization and transgenic fluorescent reporter models. We used an antisense morpholino to create a zebrafish acardiac model. For thyroid rescue experiments, BMP pathway induction in zebrafish embryos was obtained by using heatshock inducible transgenic lines.ResultsInterestingly, combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of morpholino-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development.ConclusionCollectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function.

BackgroundDefects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification and subsequent morphogenetic processes in thyroid development.MethodsTo begin to address this problem in a systematic way, we used zebrafish embryos to perform a series ofin vivophenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm and cardiovascular development using whole mountin situhybridization and transgenic fluorescent reporter models.ResultsSystematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding as well as hypoplastic, enlarged or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified BMP and FGF signaling as key factors for thyroid specification and early thyroid organogenesis, highlight the importance of low Wnt activities during early development for thyroid specification and implicate drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis.ConclusionsBy integrating the outcome of our screening efforts with previously available information from other model organisms includingXenopus, chicken and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues., SCOPUS: ar.j, info:eu-repo/semantics/published

With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Congenital hypothyroidism caused by thyroid dysgenesis (CHTD) is a common congenital disorder with a birth prevalence of 1 case in 4000 live births, and up to 8% of individuals with CHTD have co-occurring congenital heart disease. Initially we found nine patients with cardiac and thyroid congenital disorders in our cohort of 158 CHTD patients. To enrich for a rare phenotype likely to be genetically simpler, we selected three patients with a ventricular septal defect for molecular studies. Then, to assess whether rare de novo copy number variants and coding mutations in candidate genes are a source of genetic susceptibility, we used a genome-wide single-nucleotide polymorphism array and Sanger sequencing to analyze blood DNA samples from selected patients with co-occurring CHTD a congenital heart disease. We found rare variants in all three patients, and we selected Netrin-1 as the biologically most plausible contributory factor for functional studies. In zebrafish, ntn1a and ntn1b were not expressed in thyroid tissue, but ntn1a was expressed in pharyngeal arch mesenchyme, and ntn1a-deficient embryos displayed defective aortic arch artery formation and abnormal thyroid morphogenesis. The functional activity of the thyroid in ntn1a-deficient larvae was, however, preserved. Phenotypic analysis of affected zebrafish indicates that abnormal thyroid morphogenesis resulted from a lack of proper guidance exerted by the dysplastic vasculature of ntn1a-deficient embryos. Hence, careful phenotyping of patients combined with molecular and functional studies in zebrafish identify Netrin-1 as a potential shared genetic factor for cardiac and thyroid congenital defects., SCOPUS: ar.j, info:eu-repo/semantics/published

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function., info:eu-repo/semantics/published

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 microg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression.

The synthetic gestagen levonorgestrel (LNG) was previously shown to perturb thyroid hormone-dependent metamorphosis in Xenopus laevis. However, so far the mechanisms underlying the anti-metamorphic effects of LNG remained unknown. Therefore, a series of in vivo and ex vivo experiments was performed to identify potential target sites of LNG action along the pituitary-thyroid axis of X. laevis tadpoles. Prometamorphic tadpoles were treated in vivo with LNG (0.01–10 nM) for 72 h and brain-pituitary and thyroid tissue was analyzed for marker gene expression. While no treatment-related changes were observed in brain-pituitary tissue, LNG treatment readily affected thyroidal gene expression in tadpoles including decreased slc5a5 and iyd mRNA expression and a strong induction of dio2 and dio3 expression. When using an ex vivo organ explant culture approach, direct effects of LNG on both pituitary and thyroid gland gene expression were detecTable Specifically, treatment of pituitary explants with 10 nM LNG strongly stimulated dio2 expression and concurrently suppressed tshb expression. In thyroid glands, ex vivo LNG treatment induced dio2 and dio3 mRNA expression in a thyrotropin-independent manner. When thyroid explants were cultured in thyrotropin-containing media, LNG caused similar gene expression changes as seen after 72 h in vivo treatment including a very strong repression of thyrotropin-induced slc5a5 expression. Concerning the anti-thyroidal activity of LNG as seen under in vivo conditions, our ex vivo data provide clear evidence that LNG directly affects expression of genes important for thyroidal iodide handling as well as genes involved in negative feedback regulation of pituitary tshb expression.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity., The AAA+ ATPase p97 is an essential hexameric protein with multiple protein interaction partners and cellular functions. Here, the authors use interaction mapping to examine partner proteins of this large complex, and assess the effects of these proteins on the disassembly of the p97 complex.

The uptake of iodide represents the first step in thyroid hormone synthesis by thyroid follicular cells and is mediated by the sodium-iodide symporter (NIS). In mammals, expression of NIS is stimulated by TSH and transcription of the NIS gene involves regulation by the thyroid-specific transcription factors Pax8 and Nkx2.1. In this study, we examined the mRNA expression of NIS, Pax8 and Nkx2.1 in the thyroid gland of Xenopus laevis tadpoles by semi-quantitative reverse transcriptase (RT)-PCR. During spontaneous metamorphosis, NIS mRNA expression was low in premetamorphic tadpoles, increased throughout prometamorphosis, and peaked at climax stage 60. Analysis of TSH β-subunit (TSHβ) mRNA in the pituitary of the same tadpoles revealed a close temporal relationship in the expression of the two genes during metamorphosis, suggesting a regulatory role of TSH in the developmental expression of NIS. Treatment of tadpoles with goitrogenic compounds (sodium perchlorate and ethylenethiourea) increased TSHβ mRNA expression (approximately twofold) and caused thyroid gland hyperplasia, confirming that feedback along the pituitary–thyroid axis was operative. Analysis of gene expression in the thyroid gland revealed that goitrogen treatment was correlated with increased expression of NIS mRNA (~20-fold). In the thyroid gland organ culture experiments, bovine TSH (bTSH; 1 mU/ml) strongly induced NIS mRNA expression. This effect was mimicked by co-culture of thyroid glands with pituitaries from stage 58 tadpoles and by agents that increase intracellular cAMP (forskolin, dibutyryl-cAMP). In addition, it could be shown that thyroid glands of X. laevis tadpoles express Pax8 and Nkx2.1 mRNA in a developmentally regulated manner and that ex vivo treatment of thyroid glands with bTSH, forskolin, and cAMP analogs increased the expression of Pax8 and Nkx2.1 mRNA. This is the first report on developmental profiles and hormonal regulation of thyroid gland gene expression in amphibian tadpoles and, together, results reveal a critical role of TSH in the regulation of NIS mRNA expression in the thyroid gland of X. laevis tadpoles.

Effects of tetrabromobisphenol A (TBBA) on thyroid hormone (TH)-dependent morphological development and on TH-regulated biomarkers were investigated using the amphibian Xenopus laevis. Tadpoles were exposed in the Xenopus metamorphosis assay to different TBBA concentrations ranging from 2.5 to 500 microg/L for 21 days. Only the highest TBBA concentration inhibited larval development while the biomarker for negative feedback mechanism, TH receptor beta-mRNA, was not affected. Short-term exposures to TBBA, triiodothyronine (T3), and a combination of TBBA and T3 were performed to examine expression of TH-upregulated biomarkers. TBBA alone increased slightly the expression of TH receptor beta- and basic region leucin zipper transcription factor b/Zip-mRNA but inhibited their T3-induced elevation in a dose-dependent manner. Despite the fact that TBBA seems to cause only mild effects during long-term treatment on larval development, short-term exposure revealed indirect evidence that TBBA can function as a TH antagonist.

Amphibian metamorphosis represents a unique biological model to study thyroid hormone (TH) action in vivo. In this study, we examined the utility of thyroid hormone receptors alpha (TRalpha) and betaA (TRbetaA) mRNA expression patterns in Xenopus laevis tadpoles as molecular markers indicating modulation of TH action. During spontaneous metamorphosis, only moderate changes were evident for TRalpha gene expression whereas a marked up-regulation of TRbetaA mRNA occurred in hind limbs (prometamorphosis), head (late prometamorphosis), and tail tissue (metamorphic climax). Treatment of premetamorphic tadpoles with 1 nM 3,5,3'-triiodothyronine (T3) caused a rapid induction of TRbetaA mRNA in head and tail tissue within 6 to 12 h which was maintained for at least 72 h after initiation of T3 treatment. Developmental stage had a strong influence on the responsiveness of tadpole tissues to induce TRbetaA mRNA during 24 h treatment with thyroxine (0, 1, 5, 10 nM T4) or T3 (0, 1, 5, 10 nM). Premetamorphic tadpoles were highly sensitive in their response to T4 and T3 treatments, whereas sensitivity to TH was decreased in early prometamorphic tadpoles and strongly diminished in late prometamorphic tadpoles. To examine the utility of TRbetaA gene expression analysis for detection of agonistic and antagonistic effects on T3 action, mRNA expression was assessed in premetamorphic tadpoles after 48 h of treatment with the synthetic agonist GC-1 (0, 10, 50, 250 nM), the synthetic antagonist NH-3 (0, 40, 200, 1000 nM), and binary combinations of NH-3 (0, 40, 200, 1000 nM) and T3 (1 nM). All tested concentrations of GC-1 as well as the highest concentration of NH-3 caused an up-regulation of TRbetaA expression. Co-treatment with NH-3 and T3 revealed strong antagonistic effects by NH-3 on T3-induced TRbetaA mRNA up-regulation. Results of this study suggest that TRbetaA mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in X. laevis tadpoles.

Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone beta-subunit (LHbeta) and follicle-stimulating hormone beta-subunit (FSHbeta) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10(-8)M during a four weeks exposure of adults of both sexes. In general, males had a higher LHbeta mRNA level compared to females, while the mRNA expression of FSHbeta and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHbeta mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHbeta mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHbeta mRNA and FSHbeta mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHbeta mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

A partial dissociation of the HA1 subunits of influenza virus hemagglutinin (HA) is considered to be the initial step of conformational changes of the HA ectodomain leading to a membrane fusion active conformation (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, 1992, Cell 68:635–645; G. W. Kemble, D. L. Bodian, J. Rose, I. A. Wilson, and J. M. White, 1992, J. Virol. 66:4940–4950). Here, we explore a mechanism that provides an understanding of the physical and chemical basis for such dissociation and relies on two essential observations. First, based on the x-ray structure of HA from X31 (I. A. Wilson, J. J. Skehel, and D. C. Wiley, 1981, Nature 289:366–373), and by employing techniques of molecular modeling, we show that the protonation of the HA1 subunits is enhanced at the conditions known to trigger conformational changes of the HA ectodomain. Second, we found that the dependence of the calculated relative degree of protonation of the HA1 domain on temperature and pH is similar to that observed experimentally for the conformational change of HA assessed by proteinase K sensitivity. We suggest that at the pH-temperature conditions typical for the conformational change of HA and membrane fusion, dissociation of the HA1 subunits is caused by the enhanced protonation of the HA1 subunits leading to an increase in the positive net charge of these subunits and, in turn, to a weakened attraction between them.

Thyroid dysgenesis (TD) resulting from defects during embryonic thyroid development represents a major cause of congenital hypothyroidism. The pathogenetic mechanisms of TD in human newborns, however, are still poorly understood and disease-causing genetic variants have been identified in only a small percentage of TD cases. This limited understanding of the pathogenesis of TD is partly due to a lack of knowledge on how intrinsic factors and extrinsic signalling cues orchestrate the differentiation of thyroid follicular cells and the morphogenesis of thyroid tissue. Recently, embryonic stem cells and zebrafish embryos emerged as novel model systems that allow for innovative experimental approaches in order to decipher cellular and molecular mechanisms of thyroid development and to unravel pathogenic mechanisms of TD. Zebrafish embryos offer several salient properties for studies on thyroid organogenesis including rapid and external development, optical transparency, ease of breeding, relative short generation time and amenability for genome editing. In this review, we will highlight recent advances in the zebrafish toolkit to visualize cellular dynamics of organ development and discuss specific prospects of the zebrafish model for studies on vertebrate thyroid development and human congenital thyroid diseases.

The thyroid hormone (TH) system is involved in several important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility and cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDCs), Information on potential of chemicals causing THD is typically derived from animal studies. For the majority of chemicals, however, this information is either limited or unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern '-omics' technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommended a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD. Published by Elsevier Ltd.

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Among the various organs derived from foregut endoderm, the thyroid gland is unique in that major morphogenic events such as budding from foregut endoderm, descent into subpharyngeal mesenchyme and growth expansion occur in close proximity to cardiovascular tissues. To date, research on thyroid organogenesis was missing one vital tool—a transgenic model that allows to track the dynamic changes in thyroid size, shape and location relative to adjacent cardiovascular tissues in live embryos. In this study, we generated a novel transgenic zebrafish line, tg(tg:mCherry), in which robust and thyroid-specific expression of a membrane version of mCherry enables live imaging of thyroid development in embryos from budding stage throughout formation of functional thyroid follicles. By using various double transgenic models in which EGFP expression additionally labels cardiovascular structures, a high coordination was revealed between thyroid organogenesis and cardiovascular development. Early thyroid development was found to proceed in intimate contact with the distal ventricular myocardium and live imaging confirmed that thyroid budding from the pharyngeal floor is tightly coordinated with the descent of the heart. Four-dimensional imaging of live embryos by selective plane illumination microscopy and 3D-reconstruction of confocal images of stained embryos yielded novel insights into the role of specific pharyngeal vessels, such as the hypobranchial artery (HA), in guiding late thyroid expansion along the pharyngeal midline. An important role of the HA was corroborated by the detailed examination of thyroid development in various zebrafish models showing defective cardiovascular development. In combination, our results from live imaging as well es from 3D-reconstruction of thyroid development in tg(tg:mCherry) embryos provided a first dynamic view of late thyroid organogenesis in zebrafish—a critical resource for the design of future studies addressing the molecular mechanisms of these thyroid-vasculature interactions.

Environmental compounds can interfere with endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disrupters (ED), are surface waters. Thus, aquatic vertebrates, such as fish and amphibians, are most endangered. ED can adversely affect reproductive biology and the thyroid system. ED act by (anti)estrogenic and (anti)androgenic modes of action, resulting in abnormal sexual differentiation and impaired reproduction. These effects are mainly driven by direct interferences of ED with sex steroid receptors rather than indirectly by impacting synthesis and bioavailability of sex steroids, which in turn might affect the hypothalamic-pituitary-gonadal axis. Recent findings reveal that, in addition to the human-produced waste of ED, natural sources, such as parasites and decomposition of leaves, also might act as ED, markedly affecting sexual differentiation and reproduction in fish and amphibians. Although the thyroid system has essential functions in both fish and amphibians, amphibian metamorphosis has been introduced as the most sensitive model to detect thyroidal ED; no suitable fish model exists. Whereas ED may act primarily on only one specific endocrine target, all endocrine systems will eventually be deregulated as they are intimately connected to each other. The recent ecotoxicological issue of pharmaceutically active compounds (PhACs) present in the aquatic environment indicates a high potential for further endocrine modes of action on aquatic vertebrates by ED derived from PhACs, such as glucocorticoids, progestins, and beta-agonists.

The present study investigated effects of chronic treatment with corticosterone (B) on larval development of the amphibian Xenopus laevis. Tadpoles (stage 51) were treated with B (100, 500 nmol/L), 50 mg/L of the anti-thyroidal substance ethylenethiourea (ETU), and a combination of ETU (50 mg/L) plus B (500 nmol/L) to address corticosteroid effects in the presence or absence of thyroid hormones (TH). ETU-treated tadpoles displayed developmental arrest, reduced levels of TH receptor beta (TRbeta) and prolactin (PRL) mRNAs, but increased expression of thyroid-stimulating hormone (TSHbeta). Tadpoles exposed to B were characterized by asynchrony of metamorphic tissue remodeling and decreased mRNA expression of corticotropin-releasing factor (CRF). Cotreatment of ETU and B reversed the effects of ETU on PRL and TSHbeta expression but did not alter ETU effects on development and TRbeta expression. Our findings confirm that chronic treatment with high doses of corticosteroids has teratogenic effects on developing tadpoles and additionally suggest a stimulatory effect of B on gene expression of the antimetamorphic pituitary hormone PRL.

Although thyroid hormones (TH) are the primary morphogens regulating amphibian metamorphosis, other hormones including corticosteroids are known to participate in this regulation. The present study investigated effects of corticosteroids on larval development of the amphibian Xenopus laevis. Premetamorphic tadpoles (stage 51) were treated with aldosterone (ALDO; 100 nM), corticosterone (B; 10, 100, 500 nM) and dexamethasone (DEX; 10, 100, 500 nM) for 21 days and organismal responses were assessed by gross morphology determining stage development, whole body length (WBL), and hind limb length (HLL). B and DEX reduced WBL and HLL and caused abnormal development including the lack of fore limb emergence while ALDO treatment showed no significant effect. Gene expression analyses using RT-PCR revealed up-regulation of prolactin (PRL) in brain, but down-regulation of type III deiodinase in tail tissue induced by the glucocorticoids B and DEX. Additionally, stromelysin-3 transcript in tail tissue was decreased by B. ALDO at 100 nM had no effect on mRNA expression, neither in brain nor in tail tissue. These findings indicate that corticosteroids modulate TH-dependent metamorphosis by complex mechanisms that even include indirect effects triggered by increased PRL mRNA expression.

Amphibian metamorphosis represents a promising model for the identification of thyroid system-disrupting chemicals due to the pivotal role played by thyroid hormones for the initiation and regulation of metamorphosis. An important aspect of bioassay development is the identification and evaluation of sensitive and diagnostic endpoints. In this study, several morphological, histological, and molecular endpoints were evaluated for their utility to detect alterations in thyroid system function after exposure of stage 51 Xenopus laevis tadpoles to various concentrations (1.0, 2.5, 10, 25, and 50 mg/l) of the anti-thyroidal compound ethylenethiourea (ETU). Analysis of developmental stages on exposure day 20 and monitoring of time to fore limb emergence (FLE) revealed retardation and complete arrest of tadpole development at 25 mg/l and 50 mg/l ETU, respectively. Development was not affected by 1.0, 2.5, and 10 mg/l ETU. Histological alterations in the thyroid gland were observed in FLE-displaying tadpoles after exposure to 2.5, 10, and 25 mg/l ETU, as well as in developmentally arrested tadpoles exposed to 50 mg/l ETU. Prevalence and severity of histological changes increased in a concentration-dependent manner. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increased mRNA expression of the alpha- and beta-subunits of thyroid-stimulating hormone (TSHalpha, TSHbeta) in pituitary tissue of tadpoles exposed to 25 and 50 mg/l ETU. Results demonstrate the successful detection of anti-thyroidal effects of ETU in Xenopus laevis tadpoles using various endpoints and highlight the particular sensitivity of thyroid gland histology to detect thyroid system disruption in tadpoles.

A need is recognized for the development and evaluation of bioassays for detection of thyroid system-disrupting compounds. The issue of testing for thyroid disruption can be addressed by exploiting amphibian metamorphosis as a biological model. In the present study, a test protocol for a Xenopus metamorphosis assay (XEMA) was developed and its interlaboratory transferability was evaluated in an informal ring test with six laboratories participating. In the XEMA test, exposure of Xenopus laevis tadpoles was initiated at stages 48 to 50 and continued for 28 d. Development and growth of tadpoles were assessed by means of developmental stage and whole body length determinations, respectively. For initial test protocol evaluation, thyroxine (T4), and propylthiouracil (PTU) were used as positive controls for thyroid system-modulating activity, and ethylenethiourea (ETU) was used as a test compound. Exposure of tadpoles to 1 microg/L T4 produced a significant acceleration of metamorphosis whereas PTU concentrations of 75 and 100 mg/L completely inhibited metamorphosis. Five different ETU concentrations (5, 10, 25, 50, and 100 mg/L) were tested and a concentration-dependent inhibition of metamorphosis was observed. None of the compounds affected tadpole survival, and only PTU caused a slight retardation in tadpole growth. This study demonstrates that the XEMA test provides a sensitive, robust, and practical testing approach for detection of compounds with both agonistic and antagonistic effects on the thyroid system in Xenopus tadpoles.