Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution.

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.

Overlapping action of T3 and T4 during Xenopus laevis development.

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 (T3 or T4), 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.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

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.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

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 (T3) and thyroxine (T4)) 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. T3, T4, 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.

Crosstalk between Thyroid Hormone and Corticosteroid Signaling Targets Cell Proliferation in Xenopus tropicalis Tadpole Liver.

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 effects on differentiating mouse embryonic stem cells reveals unexpected impact on immune system.

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.

Thyroid and Corticosteroid Signaling in Amphibian Metamorphosis.

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.

The invention of aldosterone, how the past resurfaces in pediatric endocrinology.

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.

Are paedomorphs actual larvae?

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.

Insufficiency of Thyroid Hormone in Frog Metamorphosis and the Role of Glucocorticoids.

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.

Opposite T3 Response of ACTG1-FOS Subnetwork Differentiate Tailfin Fate in Xenopus Tadpole and Post-hatching Axolotl.

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.

De Novo Transcriptomic Approach to Study Thyroid Hormone Receptor Action in Non-mammalian Models.

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.

Chromatin Immunoprecipitation for Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues.

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.

Chromatin Interaction Analysis Using Paired-End-Tag (ChIA-PET) Sequencing in Tadpole Tissues.

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 MmeI, 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.

A novel stress hormone response gene in tadpoles of Xenopus tropicalis.

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.