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Thyroid hormones and their receptors (TRs) play critical roles during vertebrate development. One of the most dramatic developmental processes regulated by thyroid hormones is frog metamorphosis, which mimics the postembryonic (perinatal) period in mammals. Here, we review some of the findings on the developmental functions of thyroid hormones and TRs as well as their associated mechanisms of action obtained from this model system. Over two decades ago, a dual function model was proposed for TR in anuran development. During larval development, unliganded receptors recruits corepressors to repress thyroid hormone response genes to prevent premature metamorphic changes. Subsequently, when thyroid hormone levels rise, liganded receptors recruit coactivators to activate thyroid hormone response genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model and shown that it is applicable to mammalian development as well as understanding the diverse effects of thyroid hormones in normal physiology and diseases caused by thyroid hormone signaling dysfunction., (Published by Oxford University Press on behalf of the Endocrine Society 2024.)

There are many concerns about the impacts of Endocrine-Disrupting Chemicals on both wildlife and human populations. A plethora of chemicals have been shown to interfere with the Hypothalamic-Pituitary-Thyroid (HPT) axis in vertebrates. Disruption of the HPT axis is one of main endocrine criteria considered for the regulation of chemicals, along with the estrogen axis, androgen axis and steroidogenesis (EATS). In response to these concerns, the Organization for Economic Cooperation and Development (OECD) initiated the validation of test guidelines (TGs) covering the EATS modalities. Regarding thyroid activity and/or disruption assessment, three OECD TGs are validated, all of them using amphibians. To date, no OECD TGs based on fish are available for the detection of Thyroid Active Chemicals (TACs). To fill this gap, we developed a new test for the detection of TACs, the TETHYS assay (Transgenic Eleuthero-embryonic THYroid-Specific assay). This assay uses a medaka (Oryzias latipes) transgenic line Tg(tg:eGFP) expressing Green Fluorescent Protein in the thyroid follicles, under the control of the thyroglobulin promoter. This assay is performed at eleuthero-embryonic life-stages with an exposure length of 72 h. In the present study, the following reference chemicals with known thyroid hormone system mechanism of action have been tested: methimazole, sodium perchlorate, sodium tetrafluoroborate, diclofenac, iopanoic acid, sobetirome, NH-3 and 1-850. Except for the thyroid receptor antagonists, all chemicals tested were identified as thyroid active, modifying the total fluorescence and the size of the thyroid follicles. To investigate the test specificity, we tested three chemicals presumed to be inert on the HPT axis: cefuroxime, abamectin and 17α-ethinylestradiol. All were found to be inactive in the TETHYS assay. This promising New Approach Methodology can serve as a foundation for the development of a new OECD TG in the frame of regulatory assessment of chemicals for thyroid activity., Competing Interests: Declaration of competing interest Laurent Sachs reports a relationship with L'Oréal SA that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lithium is a key medication for the treatment of psychiatric disorders and is also used in various industrial applications (including battery production and recycling). Here, we review published data on the endocrine-disrupting potential of lithium, with a particular focus on the thyroid hormone system. To this end, we used PubMed and Scopus databases to search for, select and review primary research addressing human and animal health endpoints during or after lithium exposure at non-teratogenic doses. Given the key role of thyroid hormones in neurodevelopmental processes, we focused at studies of the neural effects of developmental exposure to lithium in humans and animals. Our results show that lithium meets the World Health Organization's definition of a thyroid hormone system disruptor - particularly when used at therapeutic doses. When combined with knowledge of adverse outcome pathways linking molecular initiating events targeting thyroid function and neurodevelopmental outcomes, the neurodevelopmental data reported in animal experiments prompt us to suggest that lithium influences neurodevelopment. However, we cannot rule out the involvement of additional modes of action (i.e. unrelated to the thyroid hormone system) in the described neural effects. Given the increasing use of lithium salts in new technologies, attention must be paid to this emerging pollutant - particularly with regard to its potential effects at environmental doses on the thyroid hormone system and potential consequences on the developing nervous system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T 3 or T 4 ), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tribondeau, Du Pasquier, Benchouaia, Blugeon, Buisine and Sachs.)

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed., (© 2024. The Author(s).)

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka ( Oryzias latipes ) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T 3 ) and thyroxine (T 4 )) or inhibitors (6- N -propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T 3 , T 4 , PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Thyroid hormones (TH) and glucocorticoids (GC) are involved in numerous developmental and physiological processes. The effects of individual hormones are well documented, but little is known about the joint actions of the two hormones. To decipher the crosstalk between these two hormonal pathways, we conducted a transcriptional analysis of genes regulated by TH, GC, or both hormones together in liver of Xenopus tropicalis tadpoles using RNA-Seq. Among the differentially expressed genes (DE), 70.5% were regulated by TH only, 0.87% by GC only, and 15% by crosstalk between the two hormones. Gene ontology analysis of the crosstalk-regulated genes identified terms referring to DNA replication, DNA repair, and cell-cycle regulation. Biological network analysis identified groups of genes targeted by the hormonal crosstalk and corroborated the gene ontology analysis. Specifically, we found two groups of functionally linked genes (chains) mainly composed of crosstalk-regulated hubs (highly interactive genes), and a large subnetwork centred around the crosstalk-regulated genes psmb6 and cdc7 . Most of the genes in the chains are involved in cell-cycle regulation, as are psmb6 and cdc7 , which regulate the G2/M transition. Thus, the biological action of these two hormonal pathways acting together in the liver targets cell-cycle regulation.

Tetrabromobisphenol A (TBBPA) is a potent flame retardant used in numerous appliances and a major pollutant in households and ecosystems. In vertebrates, it was shown to affect neurodevelopment, the hypothalamic-pituitary-gonadal axis and thyroid signaling, but its toxicity and modes of actions are still a matter of debate. The molecular phenotype resulting from exposure to TBBPA is only poorly described, especially at the level of transcriptome reprogramming, which further limits our understanding of its molecular toxicity. In this work, we combined functional genomics and system biology to provide a system-wide description of the transcriptomic alterations induced by TBBPA acting on differentiating mESCs, and provide potential new toxicity markers. We found that TBBPA-induced transcriptome reprogramming affect a large collection of genes loosely connected within the network of biological pathways, indicating widespread interferences on biological processes. We also found two hotspots of action: at the level of neuronal differentiation markers, and surprisingly, at the level of immune system functions, which has been largely overlooked until now. This effect is particularly strong, as terminal differentiation markers of both myeloid and lymphoid lineages are strongly reduced: the membrane T cell receptor (Cd79a, Cd79b), interleukin seven receptor (Il7r), macrophages cytokine receptor (Csf1r), monocyte chemokine receptor (Ccr2). Also, the high affinity IgE receptor (Fcer1g), a key mediator of allergic reactions, is strongly induced. Thus, the molecular imbalance induce by TBBPA may be stronger than initially realized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tribondeau, Sachs and Buisine.)

In multicellular organisms, development is based in part on the integration of communication systems. Two neuroendocrine axes, the hypothalamic-pituitary-thyroid and the hypothalamic-pituitary-adrenal/interrenal axes, are central players in orchestrating body morphogenesis. In all vertebrates, the hypothalamic-pituitary-thyroid axis controls thyroid hormone production and release, whereas the hypothalamic-pituitary-adrenal/interrenal axis regulates the production and release of corticosteroids. One of the most salient effects of thyroid hormones and corticosteroids in post-embryonic developmental processes is their critical role in metamorphosis in anuran amphibians. Metamorphosis involves modifications to the morphological and biochemical characteristics of all larval tissues to enable the transition from one life stage to the next life stage that coincides with an ecological niche switch. This transition in amphibians is an example of a widespread phenomenon among vertebrates, where thyroid hormones and corticosteroids coordinate a post-embryonic developmental transition. The review addresses the functions and interactions of thyroid hormone and corticosteroid signaling in amphibian development (metamorphosis) as well as the developmental roles of these two pathways in vertebrate evolution.

Sodium and water homeostasis are drastically modified at birth, in mammals, by the transition from aquatic life to terrestrial life. Accumulating evidence during the past ten years underscores the central role for the mineralocorticoid signaling pathway, in the fine regulation of this equilibrium, at this critical period of development. Interestingly, regarding evolution, while the mineralocorticoid receptor is expressed in fish, the appearance of its related ligand, aldosterone, coincides with terrestrial life, as it is first detected in lungfish and amphibian. Thus, aldosterone is likely one of the main hormones regulating the transition from an aquatic environment to an air environment. This review will focus on the different actors of the mineralocorticoid signaling pathway from aldosterone secretion in the adrenal gland, to mineralocorticoid receptor expression in the kidney, summarizing their regulation and roles throughout fetal and neonatal development, in the light of evolution., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis., Methods: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology., Results: We found that gene expression is mostly regulated by either T 3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks., Conclusion: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC > TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.

Amphibians display very diverse life cycles and development can be direct, where it occurs in ovo and a juvenile hatches directly, or biphasic, where an aquatic larva hatches and later undergoes metamorphosis followed by sexual maturation. In both cases, metamorphosis, corresponds to the post embryonic transition (PETr). A third strategy, only found in Urodeles, is more complex as larvae reach sexual maturity before metamorphosis, which can become accessory. The resulting paedomorphs retain their larval characters and keep their aquatic habitat. Does it mean that paedomorphs do not undergo PETr? Recent work using high throughput technologies coupled to system biology and developmental endocrinology revisited this question and provided novel datasets indicating that a paedomorph's "larval" tissue undergoes a proper developmental transition. Together with historical data, we propose that this transition is a marker of the PETr, which would be distinct from metamorphosis. This implies that (a) complex life cycles would result from the uncoupling of PETr and metamorphosis, and (b) biphasic life cycles would be a special cases where they occur simultaneously., (© 2021 American Association of Anatomists.)

Thyroid hormone (TH) is the most important hormone in frog metamorphosis, a developmental process which will not occur in the absence of TH but can be induced precociously by exogenous TH. However, such treatments including in-vitro TH treatments often do not replicate the events of natural metamorphosis in many organs, including lung, brain, blood, intestine, pancreas, tail, and skin. A potential explanation for the discrepancy between natural and TH-induced metamorphosis is the involvement of glucocorticoids (GCs). GCs are not able to advance development by themselves but can modulate the rate of developmental progress induced by TH via increased tissue sensitivity to TH. Global gene expression analyses and endocrine experiments suggest that GCs may also have direct actions required for completion of metamorphosis independent of their effects on TH signaling. Here, we provide a new review and analysis of the requirement and necessity of TH signaling in light of recent insights from gene knockout frogs. We also examine the independent and interactive roles GCs play in regulating morphological and molecular metamorphic events dependent upon TH.

Amphibian post-embryonic development and Thyroid Hormones (TH) signaling are deeply and intimately connected. In anuran amphibians, TH induce the spectacular and complex process known as metamorphosis. In paedomorphic salamanders, at similar development time, raising levels of TH fail to induce proper metamorphosis, as many "larval" tissues (e.g., gills, tailfin) are maintained. Why does the same evolutionary conserved signaling pathway leads to alternative phenotypes? We used a combination of developmental endocrinology, functional genomics and network biology to compare the transcriptional response of tailfin to TH, in the post-hatching paedormorphic Axolotl salamander and Xenopus tadpoles. We also provide a technological framework that efficiently reduces large lists of regulated genes down to a few genes of interest, which is well-suited to dissect endocrine regulations. We first show that Axolotl tailfin undergoes a strong and robust TH-dependent transcriptional response at post embryonic transition, despite the lack of visible anatomical changes. We next show that Fos and Actg1, which structure a single and dense subnetwork of cellular sensors and regulators, display opposite regulation between the two species. We finally show that TH treatments and natural variations of TH levels follow similar transcriptional dynamics. We suggest that, at the molecular level, tailfin fate correlates with the alternative transcriptional states of an fos-actg1 sub-network, which also includes transcription factors and regulators of cell fate. We propose that this subnetwork is one of the molecular switches governing the initiation of distinct TH responses, with transcriptional programs conducting alternative tailfin fate (maintenance vs. resorption) 2 weeks post-hatching.

Proper gene expression involves communication between the regulatory elements and promoters of genes. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity through DNA looping between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing is a powerful high-throughput method for detection of genome-wide chromatin interactions. Following enrichment of the chromatin complexes with a dedicated antibody, through a process of immunoprecipitation (IP), DNA fragments are end-joined with specifically designed DNA-linkers through proximity ligation. The DNA-linkers contain the binding site for the type II restriction enzyme Mme I, which cleaves 20 bp from each end of the ligated fragments, thus releasing a "paired end tag" (PET): [20 bp tag]-[linker]-[20 bp tag]. The PETs are then deep-sequenced and reads are mapped to the reference genome, revealing both binding sites, as well as remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIA-PET library construction and can be completed in 2 wk., (© 2018 Cold Spring Harbor Laboratory Press.)

Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing, is a leading high-throughput method for detection of genome wide chromatin interactions. Briefly, the method involves cross-linkage of chromatin (-DNA) fibers in cells in situ, fragmentation of the fixed chromatin-DNA complexes by sonication, followed by enrichment of the chromatin complexes with a dedicated antibody through the process of immunoprecipitation (IP). Next, application of the ChIA-PET protocol followed by deep sequencing and mapping of reads to the reference genome reveals both binding sites and remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIP sample preparation and can be completed in ∼5 d. The ChIA-PET method is detailed in an associated protocol. Because not all chromatin immunoprecipitation protocols are suitable for ChIA-PET, it is important to strictly follow this procedure before performing the ChIA-PET protocol., (© 2018 Cold Spring Harbor Laboratory Press.)

Previous work identified a transcribed locus, Str. 34945, induced by the frog stress hormone corticosterone (CORT) in Xenopus tropicalis tails. Because thyroid hormone had no influence on its expression, Str. 34945 was dubbed the first "CORT-only" gene known from tadpoles. Here, we examine the genomic annotation for this transcript, hormone specificity, time course of induction, tissue distribution, and developmental expression profile. The location of Str. 34945 on the X. tropicalis genome lies between the genes ush1g (Usher syndrome 1G) and fads6 (fatty acid desaturase 6). A blast search showed that it maps to the same region on the X. laevis genome, but no hits were found in the human genome. Using RNA-seq data and conventional reverse transcriptase PCR and sequencing, we show that Str. 34945 is part of the 3' untranslated region of ush1g. We find that CORT but not aldosterone or thyroid hormone treatment induces Str. 34945 in tadpole tails and that expression of Str. 34945 achieves maximal expression within 12-24 h of CORT treatment. Among tissues, Str. 34945 is induced to the highest degree in tail, with lesser induction in lungs, liver, and heart, and no induction in the brain or kidney. During natural metamorphosis, Str. 34945 expression in tails peaks at metamorphic climax. The role of ush1g in metamorphosis is not understood, but the specificity of its hormone response and its expression in tail make ush1g valuable as a marker of CORT-response gene induction independent of thyroid hormone., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Thyroid hormones are pleiotropic hormones involved in chordates physiology. Understanding their functions and mechanisms is also instrumental to diagnose dys-regulations and get a predictive power that can applied to medicine, ecology, etc. Today, high-throughput sequencing technologies offer the opportunity to address this issue not only in model organisms but also in non-model organisms. Here, we describe a method that makes use of RNA-seq to address differential expression analysis in non-model organism.

Although 3,5,3'-triiodothyronine (T3) is considered to be the primary bioactive thyroid hormone (TH) due to its high affinity for TH nuclear receptors (TRs), new data suggest that 3,5-diiodothyronine (T2) can also regulate transcriptional networks. To determine the functional relevance of these bioactive THs, RNA-seq analysis was conducted in the cerebellum, thalamus-pituitary and liver of tilapia treated with equimolar doses of T2 or T3. We identified a total of 169, 154 and 2863 genes that were TH-responsive (FDR < 0.05) in the tilapia cerebellum, thalamus-pituitary and liver, respectively. Among these, 130, 96 and 349 genes were uniquely regulated by T3, whereas 22, 40 and 929 were exclusively regulated by T2 under our experimental paradigm. The expression profiles in response to TH treatment were tissue-specific, and the diversity of regulated genes also resulted in a variety of different pathways being affected by T2 and T3. T2 regulated gene networks associated with cell signalling and transcriptional pathways, while T3 regulated pathways related to cell signalling, the immune system, and lipid metabolism. Overall, the present work highlights the relevance of T2 as a key bioactive hormone, and reveals some of the different functional strategies that underpin TH pleiotropy.

Thyroid hormones (TH) have been mainly associated with post-embryonic development and adult homeostasis but few studies report direct experimental evidence for TH function at very early phases of embryogenesis. We assessed the outcome of altered TH signaling on early embryogenesis using the amphibian Xenopus as a model system. Precocious exposure to the TH antagonist NH-3 or impaired thyroid receptor beta function led to severe malformations related to neurocristopathies. These include pathologies with a broad spectrum of organ dysplasias arising from defects in embryonic neural crest cell (NCC) development. We identified a specific temporal window of sensitivity that encompasses the emergence of NCCs. Although the initial steps in NCC ontogenesis appeared unaffected, their migration properties were severely compromised both in vivo and in vitro. Our data describe a role for TH signaling in NCCs migration ability and suggest severe consequences of altered TH signaling during early phases of embryonic development., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Thyroid hormone (TH) signaling comprises TH transport across cell membranes, metabolism by deiodinases, and molecular mechanisms of gene regulation. Proper TH signaling is essential for normal perinatal development, most notably for neurogenesis and fetal growth. Knowledge of perinatal TH endocrinology needs improvement to provide better treatments for premature infants and endocrine diseases during gestation and to counteract effects of endocrine disrupting chemicals. Studies in amphibians have provided major insights to understand in vivo mechanisms of TH signaling. The frog model boasts dramatic TH-dependent changes directly observable in free-living tadpoles with precise and easy experimental control of the TH response at developmental stages comparable to fetal stages in mammals. The hormones, their receptors, molecular mechanisms, and developmental roles of TH signaling are conserved to a high degree in humans and amphibians, such that with respect to developmental TH signaling "frogs are just little people that hop." The frog model is exceptionally illustrative of fundamental molecular mechanisms of in vivo TH action involving TH receptors, transcriptional cofactors, and chromatin remodeling. This review highlights the current need, recent successes, and future prospects using amphibians as a model to elucidate molecular mechanisms and functional roles of TH signaling during post-embryonic development., (© 2017 Wiley Periodicals, Inc.)

Thyroid hormone is essential for normal development in vertebrates. In amphibians, T3 controls metamorphosis by inducing tissue-specific gene regulation programs. A hallmark of T3 action is the modification of chromatin structure, which underlies changes in gene transcription. We found that mRNA for the de novo DNA methyltransferase (DNMT) dnmt3a, but not dnmt1, increased in the brain of Xenopus tadpoles during metamorphosis in parallel with plasma [T3]. Addition of T 3 to the rearing water caused a time-dependent increase in dnmt3a mRNA in tadpole brain, tail, and hind limb. By analyzing data from a genome-wide analysis of T3 receptor (TR) binding in tadpole tail, we identified several putative T3 response elements (TREs) within the dnmt3a locus. Using in vitro DNA binding, transient transfection-reporter, and chromatin immunoprecipitation assays for TRs, we identified two functional TREs at -7.1 kb and +5.1 kb relative to the dnmt3a transcription start site. Sequence alignment showed that these TREs are conserved between two related frog species, X. laevis and X. tropicalis, but not with amniotes. Our previous findings showed that this gene is directly regulated by liganded TRs in mouse brain, and whereas the two mouse TREs are conserved among Eutherian mammals, they are not conserved in Xenopus species. Thus, although T3 regulation of dnmt3a may be an ancient pathway in vertebrates, the genomic sites responsible for hormone regulation may have diverged or arisen by convergent evolution. We hypothesize that direct T 3 regulation of dnmt3a may be an important mechanism for modulating global changes in DNA methylation.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Genome-wide functional analyses require high-resolution genome assembly and annotation. We applied ChIA-PET to analyze gene regulatory networks, including 3D chromosome interactions, underlying thyroid hormone (TH) signaling in the frog Xenopus tropicalis. As the available versions of Xenopus tropicalis assembly and annotation lacked the resolution required for ChIA-PET we improve the genome assembly version 4.1 and annotations using data derived from the paired end tag (PET) sequencing technologies and approaches (e.g., DNA-PET [gPET], RNA-PET etc.). The large insert (~10 Kb, ~17 Kb) paired end DNA-PET with high throughput NGS sequencing not only significantly improved genome assembly quality, but also strongly reduced genome "fragmentation", reducing total scaffold numbers by ~60%. Next, RNA-PET technology, designed and developed for the detection of full-length transcripts and fusion mRNA in whole transcriptome studies (ENCODE consortia), was applied to capture the 5' and 3' ends of transcripts. These amendments in assembly and annotation were essential prerequisites for the ChIA-PET analysis of TH transcription regulation. Their application revealed complex regulatory configurations of target genes and the structures of the regulatory networks underlying physiological responses. Our work allowed us to improve the quality of Xenopus tropicalis genomic resources, reaching the standard required for ChIA-PET analysis of transcriptional networks. We consider that the workflow proposed offers useful conceptual and methodological guidance and can readily be applied to other non-conventional models that have low-resolution genome data.

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

Background: Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. However, the in vivo mechanism of TR action remains poorly explored., Scope of Review: Frog metamorphosis is controlled by TH and mimics the postembryonic period in mammals when high levels of TH are also required. We review here some of the findings on the developmental functions of TH and TR and the associated mechanisms obtained from this model system., Major Conclusion: A dual function model for TR in Anuran development was proposed over a decade ago. That is, unliganded TR recruits corepressors to TH response genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes. Subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model. Specifically, it has been shown that unliganded TR recruits histone deacetylase containing corepressor complexes during larval stages to control metamorphic timing, while liganded TR recruits multiple histone modifying and chromatin remodeling coactivator complexes during metamorphosis. These complexes can alter chromatin structure via nucleosome position alterations or eviction and histone modifications to contribute to the recruitment of transcriptional machinery and gene activation., General Significance: The molecular mechanisms of TR action in vivo as revealed from studies on amphibian metamorphosis are very likely applicable to mammalian development as well. These findings provide a new perspective for understanding the diverse effects of TH in normal physiology and diseases caused by TH dysfunction. This article is part of a Special Issue entitled Thyroid hormone signalling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Amphibian metamorphosis is marked by dramatic thyroid hormone (T(3))-induced changes including de novo morphogenesis, tissue remodeling, and organ resorption through programmed cell death. These changes involve cascades of gene regulation initiated by thyroid hormone (TH). TH functions by regulating gene expression through TH receptors (TR). TR are DNA-binding transcription factors that belong to the steroid hormone receptor superfamily. In the absence of ligand, TR can repress gene expression by recruiting a corepressor complex, whereas liganded TR recruits a coactivator complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving corepressors. During metamorphosis, endogenous T(3) allows TR to activate gene expression. To fully understand the diversity of T(3) effects during metamorphosis, whole genome analysis of transcriptome and mechanism of TR action should be carried out. To this end, the new sequencing technologies have dramatically changed how fundamental questions in biology are being addressed and is now making the transition from technology development to being a standard for genomic and functional genomic analysis. This review focuses on the applications of high-throughput technologies to the field of amphibian metamorphosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Amphibian metamorphosis is a well-established model for dissecting the mechanisms underlying thyroid hormone (TH) action. How the pro-hormone, T(4), the active form, T(3), the deiodinases and the nuclear receptors (TRs) contribute to metamorphosis in Xenopus has been extensively investigated. Our recent work has concentrated on two key ideas in TH signalling in Xenopus: first, that there could be active roles for both liganded and unliganded receptors, and second, that ligand availability is a determining factor orchestrating these actions and is tightly controlled in target tissues. Recently, we addressed these questions at stages preceding metamorphosis, i.e. during embryogenesis, before differentiation of a functional thyroid gland. We show that repression by unliganded TR is essential to craniofacial and eye development during early development and that at these stages all three deiodinases are active. These results open new perspectives on the potential roles of TH signalling during embryogenesis.

Stable integration of foreign DNA into the frog genome has been the purpose of several studies aimed at generating transgenic animals or producing mutations of endogenous genes. Inserting DNA into a host genome can be achieved in a number of ways. In Xenopus, different strategies have been developed which exhibit specific molecular and technical features. Although several of these technologies were also applied in various model organizms, the attributes of each method have rarely been experimentally compared. Investigators are thus confronted with a difficult choice to discriminate which method would be best suited for their applications. To gain better understanding, a transgenesis workshop was organized by the X-omics consortium. Three procedures were assessed side-by-side, and the results obtained are used to illustrate this review. In addition, a number of reagents and tools have been set up for the purpose of gene expression and functional gene analyses. This not only improves the status of Xenopus as a powerful model for developmental studies, but also renders it suitable for sophisticated genetic approaches. Twenty years after the first reported transgenic Xenopus, we review the state of the art of transgenic research, focusing on the new perspectives in performing genetic studies in this species.

Thyroid hormone (T(3)) induces gene regulation programs necessary for tadpole metamorphosis. Among the earliest responses to T(3) are the up-regulation of T(3) receptor beta (TRbeta; autoinduction) and BTEB1 (basic transcription element-binding protein 1). BTEB1 is a member of the Krüppel family of transcription factors that bind to GC-rich regions in gene promoters. The proximal promoter of the Xenopus laevis TrbetaA gene has seven GC-rich sequences, which led us to hypothesize that BTEB1 binds to and regulates TrbetaA. In tadpoles and the frog fibroblast-derived cell line XTC-2, T(3) up-regulated Bteb1 mRNA with faster kinetics than TrbetaA, and Bteb1 mRNA correlated with increased BTEB1 protein expression. BTEB1 bound to GC-rich sequences in the proximal TrbetaA promoter in vitro. By using chromatin immunoprecipitation assay, we show that BTEB1 associates with the TrbetaA promoter in vivo in a T(3) and developmental stage-dependent manner. Induced expression of BTEB1 in XTC-2 cells caused accelerated and enhanced autoinduction of the TrbetaA gene. This enhancement was lost in N-terminal truncated mutants of BTEB1. However, point mutations in the zinc fingers of BTEB1 that destroyed DNA binding did not alter the activity of the protein on TrbetaA autoinduction, suggesting that BTEB1 can function in this regard through protein-protein interactions. Our findings support the hypothesis that BTEB1 associates with the TrbetaA promoter in vivo and enhances autoinduction, but this action does not depend on its DNA binding activity. Cooperation among the protein products of immediate early genes may be a common mechanism for driving developmental signaling pathways.

Amphibian metamorphosis is an excellent model to study the diverse effects of thyroid hormones (TH). TH modulate target gene expression via thyroid hormone receptors (TR). Generally, unliganded TR repress transcription, whereas liganded TR activate transcription. During metamorphosis, these dual effects of TR are evident. Moreover, we show that gene specific response to TH can underline the multiple effects of TH. Finally, studies of unliganded-thyroid hormone receptor function reveal a physiological role in eye development.

Xenopus is a well proven model for a wide variety of developmental studies, including cell lineage. Cell lineage in Xenopus has largely been addressed by injection of tracer molecules or by micro-dissection elimination of blastomeres. Here we describe a genetic method for cell ablation based on the use of tBid, a direct activator of the mitochondrial apoptotic pathway. In mammalian cells, cross-talk between the main apoptotic pathways (the mitochondrial and the death domain protein pathways) involve the pro-death protein BID, the active form of which, tBID, results from protease truncation and translocation to mitochondria. In transgenic Xenopus, restricting tBID expression to the lens-forming cells enables the specific ablation of the lens without affecting the development of other eye structures. Thus, overexpression of tBid can be used in vivo as a tool to eliminate a defined cell population by apoptosis in a developing organism and to evaluate the degree of autonomy or the inductive effects of a specific tissue during embryonic development.

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.

Transcriptional control of hypothalamic thyrotropin-releasing hormone (TRH) integrates central regulation of the hypothalamo-hypophyseal-thyroid axis and hence thyroid hormone (triiodothyronine (T(3))) homeostasis. The two beta thyroid hormone receptors, TRbeta1 and TRbeta2, contribute to T(3) feedback on TRH, with TRbeta1 having a more important role in the activation of TRH transcription. How TRbeta1 fulfils its role in activating TRH gene transcription is unknown. By using a yeast two-hybrid screening of a mouse hypothalamic complementary DNA library, we identified a novel partner for TRbeta1, hepatitis virus B X-associated protein 2 (XAP2), a protein first identified as a co-chaperone protein. TR-XAP2 interactions were TR isoform specific, being observed only with TRbeta1, and were enhanced by T(3) both in yeast and mammalian cells. Furthermore, small inhibitory RNA-mediated knockdown of XAP2 in vitro affected the stability of TRbeta1. In vivo, siXAP2 abrogated specifically TRbeta1-mediated (but not TRbeta2) activation of hypothalamic TRH transcription. This study provides the first in vivo demonstration of a regulatory, physiological role for XAP2.

Elimination of tadpole organs during Xenopus metamorphosis is largely achieved through apoptosis, and recent evidence suggest involvement of the mitochondrial death route and bax-initiated caspase-3 and -9 deployment. However, events upstream of the activation of Bax are unknown. In other models, proteins of the BH3-only group such as BID are known to assure this function. We show that Xenopus bid transcript levels increase at metamorphosis in larval cells destined to disappear. This increase correlates with an abrupt rise in Caspase-2 and -8 mRNA levels and an enhanced activity of Caspase-2 and -8. In BIDGFP transgenic animal's tail regression is accelerated. The cleavage of BIDGFP fusion protein during natural or T(3)-induced metamorphosis was specifically inhibited by caspase-8 inhibitors. Our results show that tail regression at metamorphosis implicates an apoptotic pathway inducible by T(3) hormone in an organ autonomous manner and involving the cell death executioners BID and Caspases-2 and -8.

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., (Copyright (c) 2004 Wiley-Liss, Inc.)

The biologic role of hormonal activation of nuclear receptors is well established. Only recently, however, has the biologic significance of repression begun to be appreciated. Amphibian metamorphosis is marked by dramatic thyroid hormone induced changes, including de novo morphogenesis, tissue remodeling, and organ resorption through programmed cell death. These changes involve cascades of gene regulation initiated by 3,5,3'-triiodothyronine (T(3)). T(3) functions by regulating gene expression through thyroid hormone receptor (TR). TRs are DNA-binding transcription factors that belong to the steroid hormone receptor superfamily. In the absence of a ligand, TRs can repress gene expression by recruiting corepressor complexes, whereas liganded TRs recruit coactivator complexes for gene activation. Corepressor and coactivator complexes induce chromatin remodeling to mediate TR regulation of transcription. The mechanisms of TR action permit a dual function for TRs during development. In premetamorphic tadpoles, when TRs are expressed and T(3) levels are barely detectable, unliganded TRs repress transcription through corepressor recruitment. This TR-mediated repression of target genes is critical for proper larval development, allowing tadpole growth and acquisition of metamorphic competence. In contrast, during metamorphosis, endogenous T(3) causes TRs to activate gene expression, leading to tadpole transformation. Several results also support a role for corepressors during metamorphosis. Corepressor targeted functions, however, are still speculative but may again involve TRs. The requirement of active gene repression at different stages during amphibian development establishes an important biologic role for corepressors.

Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; tri-iodothyronine), apo-TRs associate with co-repressors to repress transcription, whereas in the presence of T3, holo-TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3-dependent amphibian metamorphosis to analyse how TRs interact with particular co-regulators to differentially regulate gene expression during development. Using chromatin immuno-precipitation to study tissue from pre-metamorphic tadpoles, we found that TRs are physically associated with T3-responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3-response genes. Most importantly, we show that individual T3-response genes have distinct co-regulator requirements, the T3-dependent co-repressor-to-coactivator switch being gene-specific for both co-regulator categories.

Transcriptional activation from chromatin by nuclear receptors (NRs) requires multiple cofactors including CBP/p300, SWI/SNF and Mediator. How NRs recruit these multiple cofactors is not clear. Here we show that activation by androgen receptor and thyroid hormone receptor is associated with the promoter targeting of SRC family members, p300, SWI/SNF and the Mediator complex. We show that recruitment of SWI/SNF leads to chromatin remodeling with altered DNA topology, and that both SWI/SNF and p300 histone acetylase activity are required for hormone-dependent activation. Importantly, we show that both the SWI/SNF and Mediator complexes can be targeted to chromatin by p300, which itself is recruited through interaction with SRC coactivators. Furthermore, histone acetylation by CBP/p300 facilitates the recruitment of SWI/SNF and Mediator. Thus, our data indicate that multiple cofactors required for activation are not all recruited through their direct interactions with NRs and underscore a role of cofactor-cofactor interaction and histone modification in coordinating the recruitment of multiple cofactors.