91 research outputs found
Répression transcriptionnelle du gène TRH
Les hormones thyroïdiennes (HT : T3, T4) exerçant des effets pléiotropes chez les vertébrés, leur synthèse et leur sécrétion doivent être finement contrôlées. Elles agissent elles-mêmes sur leur production, par un système de rétrocontrôle négatif de l’expression des gènes hypothalamique TRH et hypophysaire TSH. Les fondements moléculaires de cette répression transcriptionnelle des gènes TRH et TSH par l’hormone T3, forme biologiquement la plus active des HT, restent méconnus. Certaines caractéristiques de cette régulation commencent toutefois à être identifiées, notamment le rôle spécifique des isoformes TRβ (versus TRα) des récepteurs des HT. La spécificité fonctionnelle de ces isoformes résiderait principalement dans leur extrémité aminoterminale, qui permettrait une interaction différentielle avec certains comodulateurs. L’objectif, aujourd’hui, est de caractériser ces comodulateurs et d’analyser leur contribution à la régulation transcriptionnelle du gène TRH par l’hormone T3.The synthesis and secretion of thyroid hormones (TH: T3, T4) must be strictly regulated. TH act on their own production via a negative feedback system. The synthesis of thyrotropin-releasing hormone (TRH), produced in the hypothalamus, and thyrotropin (TSH) in the pituitary is inhibited at the transcriptional level by TH. TRH and TSH stimulate production of TH. An outstanding, still open, question is the molecular basis of T3-dependent transcription repression of TRH and TSH genes. However, some regulatory components have been identified, with the β-TH receptor (TRβ) playing a specific regulatory role (versus TRα) in the negative feedback effects of T3 on production of TRH and TSH. Moreover, the N-terminus of TRβ is known to be a key element in this regulation. A hypothesis to explain this isoform specificity could be that TRβ and TRα interact differentially with transcriptional comodulators. Thus, it is critical to characterize these comodulators and to analyse their contribution to the transcription regulation of TRH
A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain
RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor α1 (TRα1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain
A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Xenopus Tadpoles
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, T(3)) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, T(3) 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 T(3) 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 T(3)-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pre-treatment protocol that induced competence to respond to T(3) and thus to assess T(3) effects and T(3) disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak (10(−12) M) pulse of T(3) 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
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
Exploring nervous system transcriptomes during embryogenesis and metamorphosis in Xenopus tropicalis using EST analysis
<p>Abstract</p> <p>Background</p> <p>The western African clawed frog <it>Xenopus tropicalis </it>is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as <it>Xenopus laevis </it>but is diploid and has a smaller genome of 1.7 Gbp. Therefore <it>X. tropicalis </it>is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in <it>X. tropicalis</it>.</p> <p>Results</p> <p>A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers.</p> <p>Conclusion</p> <p>These cDNA sequences permit <it>in silico </it>cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study <it>X. tropicalis </it>biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.</p
Live imaging of targeted cell ablation in Xenopus:a new model to study demyelination and repair
Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the central nervous system (CNS). In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the E. coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous pro-drug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair
Removing critical gaps in chemical test methods by developing new assays for the identification of thyroid hormone system-disrupting chemicals—the athena project
The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood–brain and blood–placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation
Statement on Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment
The number of anthropogenic chemicals, manufactured, by-products, metabolites and abiotically formed transformation products, counts to hundreds of thousands, at present. Thus, humans and wildlife are exposed to complex mixtures, never one chemical at a time and rarely with only one dominating effect. Hence there is an urgent need to develop strategies on how exposure to multiple hazardous chemicals and the combination of their effects can be assessed. A workshop, “Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment” was organized in May 2018 together with Joint Research Center in Ispra, EU-funded research projects and Commission Services and relevant EU agencies. This forum for researchers and policy-makers was created to discuss and identify gaps in risk assessment and governance of chemical mixtures as well as to discuss state of the art science and future research needs. Based on the presentations and discussions at this workshop we want to bring forward the following Key Messages:
We are at a turning point: multiple exposures and their combined effects require better management to protect public health and the environment from hazardous chemical mixtures.
Regulatory initiatives should be launched to investigate the opportunities for all relevant regulatory frameworks to include prospective mixture risk assessment and consider combined exposures to (real-life) chemical mixtures to humans and wildlife, across sectors.
Precautionary approaches and intermediate measures (e.g. Mixture Assessment Factor) can already be applied, although, definitive mixture risk assessments cannot be routinely conducted due to significant knowledge and data gaps.
A European strategy needs to be set, through stakeholder engagement, for the governance of combined exposure to multiple chemicals and mixtures. The strategy would include research aimed at scientific advancement in mechanistic understanding and modelling techniques, as well as research to address regulatory and policy needs. Without such a clear strategy, specific objectives and common priorities, research, and policies to address mixtures will likely remain scattered and insufficient
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
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