Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Development and metamorphosis in frogs deficient in the thyroid hormone transporter MCT8.

Precisely regulated thyroid hormone (TH) signaling within tissues during frog metamorphosis gives rise to the organism-wide coordination of developmental events among organs required for survival. This TH signaling is controlled by multiple cellular mechanisms, including TH transport across the plasma membrane. A highly specific TH transporter has been identified, namely monocarboxylate transporter 8 (MCT8), which facilitates uptake and efflux of TH and is differentially and dynamically expressed among tissues during metamorphosis. We hypothesized that loss of MCT8 would alter tissue sensitivity to TH and affect the timing of tissue transformation. To address this, we used CRISPR/Cas9 to introduce frameshift mutations inslc16a2, the gene encoding MCT8, inXenopus laevis. We produced homozygous mutant tadpoles with a 29-bp mutation in the l-chromosome and a 20-bp mutation in the S-chromosome. We found that MCT8 mutants survive metamorphosis with normal growth and development of external morphology throughout the larval period. Consistent with this result, the expression of the pituitary hormone regulating TH plasma levels (tshb) was similar among genotypes as was TH response gene expression in brain at metamorphic climax. Further, delayed initiation of limb outgrowth during natural metamorphosis and reduced hindlimb and tail TH sensitivity were not observed in MCT8 mutants. In sum, we did not observe an effect on TH-dependent development in MCT8 mutants, suggesting compensatory TH transport occurs in tadpole tissues, as seen in most tissues in all model organisms examined.

Glucocorticoid receptor mediates corticosterone-thyroid hormone synergy essential for metamorphosis in Xenopus tropicalis tadpoles.

In all vertebrates, thyroid hormone (TH) is critical for normal growth and development. In amphibians, corticosterone (CORT) has no action to advance development by itself but can accelerate development induced by TH. CORT accomplishes this acceleration by increasing tissue sensitivity and responsivity to TH. However, the receptor through which CORT acts to affect TH signaling is not known. To examine the role of the glucocorticoid receptor (GR), GR knockout tadpoles and wild-type tadpoles treated with the GR antagonist, RU486, were exposed to exogenous TH and/or CORT then assayed for gene expression and morphology. We found that levels of the response genes klf9 and thrb induced by TH and associated changes in morphology were decreased in GR knockout tadpoles compared to wild-type tadpoles, suggesting that GR signaling contributes to tissue responsivity to TH. To directly examine the role of GR in TH signaling, we co-treated tadpoles with TH and CORT and found that the TH response gene, thrb, was induced significantly beyond the level induced by TH alone in wild-type tadpoles but not in GR knockout tadpoles or wild-type tadpoles treated with RU486. Similarly, tail and gill resorption was greater in tadpoles treated with CORT plus TH compared to TH alone in wild-type tadpoles but not in tadpoles with impaired GR signaling. Surprisingly, even though GR knockout tadpoles die at metamorphosis, treatment with TH alone enabled their survival. These results demonstrate that signaling through GR is responsible for enhancing TH signaling and is essential for the completion of metamorphosis.

Impaired negative feedback and death following acute stress in glucocorticoid receptor knockout Xenopus tropicalis tadpoles.

Blood glucocorticoid levels are regulated by the hypothalamo-pituitary-adrenal/interrenal axis (HPA axis in mammals, HPI axis in amphibians), and negative feedback by glucocorticoid signaling is a key player in that regulation. Glucocorticoid and mineralocorticoid receptors (GR and MR) mediate negative feedback in mammals, but little is known about nuclear receptor-mediated feedback in amphibians. Because amphibians have only one corticosteroidogenic cell type responsible for glucocorticoid and mineralocorticoid production, we hypothesized that GR knockout (GRKO) tadpoles have elevated levels of glucocorticoids and mineralocorticoids as well as axis components regulating their production. We also examined the response to stress and potential for increased aldosterone signaling in GRKO tadpoles. We found that GRKO tadpoles have severe hyperactivity of the HPI axis, namely high mRNA expression levels of pomc, cyp17a1, cyp21a2, cyp11b2, and star, and high tissue content of corticosterone, aldosterone, 17-hydroxyprogesterone, 21-deoxycortisol, and progesterone. Such aberrant HPI activity was accompanied by reduced survival after acute temperature shock and shaking stress. Like mammalian models of HPA hyperactivity, GRKO tadpoles have high MR mRNA expression levels in brain, kidney, heart, and skin and high levels of the inflammatory cytokine tnf-α and the profibrotic factor tgf-ß in kidneys. This study showed GR is critical for negative feedback to the amphibian HPI axis and for survival from acute stressors. This study also showed GRKO tadpoles exhibit altered expression/overproduction of regulators of salt-water homeostasis and associated biomarkers of kidney disease.

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.

Structural Investigation of Quaternary Layered Oxides upon Na-Ion Deinsertion.

Na-ion batteries are emerging alternatives to Li-ion chemistries for large-scale energy storage applications. Quaternary layered oxide Na0.76Mn0.5Ni0.3Fe0.1Mg0.1O2 offers outstanding electrochemical performance in Na-ion batteries compared to pure-phase layered oxides because of the synergistic effect of the P/O-phase mixing. The material is indeed constituted by a mixture of P3, P2, and O3 phases, and a newly identified Na-free phase, i.e., nickel magnesium oxide phase, which improves heat removal and enhances the electrochemical performance. Herein, we structurally investigate, through synchrotron-radiation X-ray diffraction, the modifications occurring after full desodiation, detailing the material structural rearrangement upon Na removal and revealing the effect of two different charging protocols, i.e., constant current (CC) and constant current-constant voltage (CCCV). While the Na-free phase is electrochemically inactive, likely helping in homogenization of the thermal gradient in the electrode during cycling, O-P intergrown phases appear during the extraction of Na ions from interslab layers, and they are dependent on the desodiation level. The application of a constant voltage step at the end of the galvanostatic charge is responsible for a shortening of the interslab distance and a significant volume contraction (-11.9%).

Stem cell development involves divergent thyroid hormone receptor subtype expression and epigenetic modifications in the Xenopus metamorphosing intestine.

In the intestine during metamorphosis of the frog Xenopus laevis, most of the larval epithelial cells are induced to undergo apoptosis by thyroid hormone (TH), and under continued TH action, the remaining epithelial cells dedifferentiate into stem cells (SCs), which then newly generate an adult epithelium analogous to the mammalian intestinal epithelium. Previously, we have shown that the precursors of the SCs that exist in the larval epithelium as differentiated absorptive cells specifically express receptor tyrosine kinase-like orphan receptor 2 (Ror2). By using Ror2 as a marker, we have immunohistochemically shown here that these SC precursors, but not the larval epithelial cells destined to die by apoptosis, express TH receptor α (TRα). Upon initiation of TH-dependent remodeling, TRα expression remains restricted to the SCs as well as proliferating adult epithelial primordia derived from them. As intestinal folds form, TRα expression becomes localized in the trough of the folds where the SCs reside. In contrast, TRß expression is transiently up-regulated in the entire intestine concomitantly with the increase of endogenous TH levels and is most highly expressed in the developing adult epithelial primordia. Moreover, we have shown here that global histone H4 acetylation is enhanced in the SC precursors and adult primordia including the SCs, while tri-methylation of histone H3 lysine 27 is lacking in those cells during metamorphosis. Our results strongly suggest distinct roles of TRα and TRß in the intestinal larval-to-adult remodeling, involving distinctive epigenetic modifications in the SC lineage.

Glucocorticoid receptor is required for survival through metamorphosis in the frog Xenopus tropicalis.

Stress hormones, also known as glucocorticoids, are critical for survival at birth in mammals due at least in part to their importance in lung maturation. However, because air breathing is not always required for amphibian survival and because stress hormones have no known developmental impact except to modulate the developmental actions of thyroid hormone (TH), the requirement for stress hormone signaling during metamorphosis is not well understoodi. Here, we produced a glucocorticoid receptor knockout (GRKO) Xenopus line with a frameshift mutation in the first exon of the glucocorticoid receptor. Induction by exogenous corticosterone (CORT, the frog stress hormone) of the CORT response genes, klf9 (Krüppel-like factor 9, also regulated by TH) and ush1g (Usher's syndrome 1G), was completely abrogated in GRKO tadpoles. Surprisingly, GRKO tadpoles developed faster than wild-type tadpoles until forelimb emergence and then developed more slowly until their death at the climax of metamorphosis. Growth rate was not affected in GRKO tadpoles, but they achieved a smaller maximum size. Gene expression analysis of the TH response genes, thrb (TH receptor beta) and klf9 showed reduced expression in the tail at metamorphic climax consistent with the reduced development rate. These results indicate that glucocorticoid receptor is required for survival through metamorphosis and support dual roles for GR signaling in control of developmental rate.

Novel vectors for functional interrogation of Xenopus ORFeome coding sequences.

The current Xenopus ORFeome contains ~10,250 validated, full-length cDNA sequences without stop codons from Xenopus laevis and ~3,970 from Xenopus tropicalis cloned into Gateway-compatible entry vectors. To increase the utility of the ORFeome, we have constructed the Gateway-compatible destination vectors pDXTP and pDXTR, which in combination can control the spatial and temporal expression of any open reading frame (ORF). pDXTP receives a promoter/enhancer of interest, which controls the spatial expression of a doxycycline-inducible transcription factor rtTA. pDXTR receives an ORF of interest, which is controlled by a tetracycline response element enabling temporal control of ORF expression via rtTA activation by simple addition of doxycycline to the rearing water at any desired time point. These vectors can be integrated into the genome via well-established microinjection-based SceI, tol2, or phi-C31 transgenesis procedures and contain fluorescence reporters to confirm transgene integration. Cell-autonomous verification of ORF expression occurs via red nuclear fluorescence due to an mCherry-histone H2B fusion protein that is cleaved from the ORF during translation. Function of all essential features of pDXTP and pDXTR has been experimentally validated. pDXTP and pDXTR provide flexible molecular cloning and transgenesis options to accomplish tissue-specific inducible control of ORF expression in transgenic Xenopus.

Dual function model revised by thyroid hormone receptor alpha knockout frogs.

All vertebrates require thyroid hormone (TH) for normal growth and development. Plasma TH enters cells and alters gene expression via nuclear receptors TRα and TRß. In-vitro studies showed that TRs function as repressors of TH-inducible genes in the absence of TH and as activators of those same genes in the presence of TH. A dual function model was proposed to harmonize these molecular TR actions with the dynamic expression of TRs and peak in production of TH experienced during development. Conclusive tests of the repression activity of TRs early in development as predicted by the model awaited gene knockout technology targeting TRα. At the molecular level, active repression of genes involved in metamorphosis by TRα in the absence of TH was confirmed in whole bodies and intestine from TRα knockout studies. As a consequence of this reduced repression in TRα knockout animals, initiation of limb morphogenesis occurs precociously. However, subsequent limb development is retarded during rising plasma TH levels due to reduced TR-dependent responsivity to TH. In contrast to the limbs, intestine remodeling is delayed by one to two developmental stages in TRα knockout animals, despite de-repressed levels of TH-induced genes during premetamorphosis. Surprisingly, in the absence of TRα, hind limbs do not require gene induction by TH signaling to complete morphological growth and development, which is contrary to prediction by the dual function model. Full evaluation of the dual function model for all organs awaits the production of TRα and TRß double knockout frogs.

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.

Frogs model man: In vivo thyroid hormone signaling during development.

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.

Clinical Utility of the 12-Gene DCIS Score Assay: Impact on Radiotherapy Recommendations for Patients with Ductal Carcinoma In Situ.

OBJECTIVE: The aim of this study was to determine the impact of the results of the 12-gene DCIS Score assay on (i) radiotherapy recommendations for patients with pure ductal carcinoma in situ (DCIS) following breast-conserving surgery (BCS), and (ii) patient decisional conflict and state anxiety. METHODS: Thirteen sites across the US enrolled patients (March 2014-August 2015) with pure DCIS undergoing BCS. Prospectively collected data included clinicopathologic factors, physician estimates of local recurrence risk, DCIS Score results, and pre-/post-assay radiotherapy recommendations for each patient made by a surgeon and a radiation oncologist. Patients completed pre-/post-assay decisional conflict scale and state-trait anxiety inventory instruments. RESULTS: The analysis cohort included 127 patients: median age 60 years, 80 % postmenopausal, median size 8 mm (39 % ≤5 mm), 70 % grade 1/2, 88 % estrogen receptor-positive, 75 % progesterone receptor-positive, 54 % with comedo necrosis, and 18 % multifocal. Sixty-six percent of patients had low DCIS Score results, 20 % had intermediate DCIS Score results, and 14 % had high DCIS Score results; the median result was 21 (range 0-84). Pre-assay, surgeons and radiation oncologists recommended radiotherapy for 70.9 and 72.4 % of patients, respectively. Post-assay, 26.4 % of overall recommendations changed, including 30.7 and 22.0 % of recommendations by surgeons and radiation oncologists, respectively. Among patients with confirmed completed questionnaires (n = 32), decision conflict (p = 0.004) and state anxiety (p = 0.042) decreased significantly from pre- to post-assay. CONCLUSIONS: Individualized risk estimates from the DCIS Score assay provide valuable information to physicians and patients. Post-assay, in response to DCIS Score results, surgeons changed treatment recommendations more often than radiation oncologists. Further investigation is needed to better understand how such treatment changes may affect clinical outcomes.

In-vivo regulation of Krüppel-like factor 9 by corticosteroids and their receptors across tissues in tadpoles of Xenopus tropicalis.

Corticosteroids are critical for normal development and for mediating effects of stress during development in all vertebrates. Even though gene knockout studies in mouse and zebrafish have identified a number of developmental roles of corticosteroids and their receptors, the numerous pleiotropic actions of these hormones affecting various aspects of development are understudied. For the most part, neither the endogenous hormone(s) nor their receptor(s) regulating developmental processes during natural development have been determined. Here, we address this issue by elucidating the endogenous regulation of the transcription factor Krüppel-like factor 9 (klf9) across tissues during development by corticosteroid hormones (aldosterone and corticosterone) and their nuclear receptors (type-I and type-II receptors). First, we measured the developmental expression profiles of klf9 and type-I and type-II corticosteroid receptors in key target tissues, brain, lungs, and tail, during larval and metamorphic stages in Xenopus tropicalis. We also studied the corticosteroid regulation of klf9 in these tissues in-vivo using exogenous hormone treatments and receptor antagonists. Klf9 and the corticosteroid receptors were expressed in each tissue and significantly increased in expression reaching a peak at metamorphic climax, except for the type-II receptor in brain and tail whose expression did not change significantly across stages. Both corticosteroid hormones induced klf9 in each tissue, although aldosterone required a five times higher dose than corticosterone to cause a significant induction. The upregulation of klf9 by both corticosteroids was completely blocked by the use of the type-II receptor antagonist RU486 and not the type-I receptor antagonist spironolactone. These results are consistent with previous in-vitro studies and indicate for the first time in-vivo that corticosteroid regulation of klf9 occurs exclusively via corticosterone and type-II receptor interaction across tissues.

More similar than you think: Frog metamorphosis as a model of human perinatal endocrinology.

Hormonal control of development during the human perinatal period is critically important and complex with multiple hormones regulating fetal growth, brain development, and organ maturation in preparation for birth. Genetic and environmental perturbations of such hormonal control may cause irreversible morphological and physiological impairments and may also predispose individuals to diseases of adulthood, including diabetes and cardiovascular disease. Endocrine and molecular mechanisms that regulate perinatal development and that underlie the connections between early life events and adult diseases are not well elucidated. Such mechanisms are difficult to study in uterus-enclosed mammalian embryos because of confounding maternal effects. To elucidate mechanisms of developmental endocrinology in the perinatal period, Xenopus laevis the African clawed frog is a valuable vertebrate model. Frogs and humans have identical hormones which peak at birth and metamorphosis, have conserved hormone receptors and mechanisms of gene regulation, and have comparable roles for hormones in many target organs. Study of molecular and endocrine mechanisms of hormone-dependent development in frogs is advantageous because an extended free-living larval period followed by metamorphosis (1) is independent of maternal endocrine influence, (2) exhibits dramatic yet conserved developmental effects induced by thyroid and glucocorticoid hormones, and (3) begins at a developmental stage with naturally undetectable hormone levels, thereby facilitating endocrine manipulation and interpretation of results. This review highlights the utility of frog metamorphosis to elucidate molecular and endocrine actions, hormone interactions, and endocrine disruption, especially with respect to thyroid hormone. Knowledge from the frog model is expected to provide fundamental insights to aid medical understanding of endocrine disease, stress, and endocrine disruption affecting the perinatal period in humans.

Regulation of thyroid hormone-induced development in vivo by thyroid hormone transporters and cytosolic binding proteins.

Differential tissue sensitivity/responsivity to hormones can explain developmental asynchrony among hormone-dependent events despite equivalent exposure of each tissue to circulating hormone levels. A dramatic vertebrate example is during frog metamorphosis, where transformation of the hind limb, brain, intestine, liver, and tail are completely dependent on thyroid hormone (TH) but occurs asynchronously during development. TH transporters (THTs) and cytosolic TH binding proteins (CTHBPs) have been proposed to affect the timing of tissue transformation based on expression profiles and in vitro studies, but they have not been previously tested in vivo. We used a combination of expression pattern, relative expression level, and in vivo functional analysis to evaluate the potential for THTs (LAT1, OATP1c1, and MCT8) and CTHBPs (PKM2, CRYM, and ALDH1) to control the timing of TH-dependent development. Quantitative PCR analysis revealed complex expression profiles of THTs and CTHBPs with respect to developmental stage, tissue, and TH receptor ß (TRß) expression. For some tissues, the timing of tissue transformation was associated with a peak in the expression of some THTs or CTHBPs. An in vivo overexpression assay by tail muscle injection showed LAT1, PKM2, and CRYM increased TH-dependent tail muscle cell disappearance. Co-overexpression of MCT8 and CRYM had a synergistic effect on cell disappearance. Our data show that each tissue examined has a unique developmental expression profile of THTs and CTHBPs and provide direct in vivo evidence that the ones tested are capable of affecting the timing of developmental responses to TH.

The current clinical value of the DCIS Score.

The management of ductal carcinoma in situ (DCIS) can be controversial. Widespread adoption of mammographic screening has made DCIS a more frequent diagnosis, and increasingly smaller, lower-grade lesions are being detected. DCIS is commonly treated with breast-conserving surgery and radiation. However, there is greater recognition that acceptable cancer control outcomes can be achieved for some patients with breast-conserving surgery alone, with radiotherapy reserved for those at higher risk of in-breast recurrence. The primary clinical dilemma is that there are currently no reliable clinicopathologic features that accurately predict which patients will have a recurrence, but risk stratification is an area of active research. Molecular profiling has the potential to assess recurrence risk based on the individual patient's tumor biology and guide treatment decisions. The DCIS Score is a 12-gene assay intended to support personalized treatment planning for patients with DCIS following local excision. It provides information on local failure risk independent of traditional clinicopathologic features. Our group of expert breast surgeons and radiation oncologists met in December 2013 at the San Antonio Breast Cancer Symposium to discuss current controversies in DCIS management and determine the potential value of the DCIS Score in managing these situations. We concluded that the DCIS Score provides clinically relevant information about personal risk that can guide patient discussions and facilitate shared decision making.

Corticosteroid signaling in frog metamorphosis.

Stress in fetal and larval life can impact later health and fitness in humans and wildlife. Long-term effects of early life stress are mediated by altered stress physiology induced during the process of relaying environmental effects on development. Amphibian metamorphosis has been an important model system to study the role of hormones in development in an environmental context. Thyroid hormone (TH) is necessary and sufficient to initiate the dramatic morphological and physiological changes of metamorphosis, but TH alone is insufficient to complete metamorphosis. Other hormones, importantly corticosteroid hormones (CSs), influence the timing and nature of post-embryonic development. Stressors or treatments with CSs delay or accelerate metamorphic change, depending on the developmental stage of treatment. Also, TH and CSs have synergistic, antagonistic, and independent effects on gene regulation. Importantly, the identity of the endogenous corticosteroid hormone or receptor underlying any gene induction or remodeling event has not been determined. Levels of both CSs, corticosterone and aldosterone, peak at metamorphic climax, and the corticosteroid receptors, glucocorticoid and mineralocorticoid receptors, have wide expression distribution among tadpole tissues. Conclusive experiments to identify the endogenous players have been elusive due to difficulties in experimental control of corticosteroid production and signaling. Current data are consistent with the hypothesis that the two CSs and their receptors serve largely overlapping functions in regulating metamorphosis and synergy with TH. Knowledge of the endogenous players is critical to understanding the basic mechanisms and significance of corticosteroid action in regulating post-embryonic development in environmental contexts.

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.

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis).

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.