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.

Minireview: Glucocorticoid-Leptin Crosstalk: Role of Glucocorticoid-Leptin Counterregulation in Metabolic Homeostasis and Normal Development.

Glucocorticoids and leptin are two important hormones that regulate metabolic homeostasis by controlling appetite and energy expenditure in adult mammals. Also, glucocorticoids and leptin strongly counterregulate each other, such that chronic stress-induced glucocorticoids upregulate the production of leptin and leptin suppresses glucocorticoid production directly via action on endocrine organs and indirectly via action on food intake. Altered glucocorticoid or leptin levels during development can impair organ development and increase the risk of chronic diseases in adults, but there are limited studies depicting the significance of glucocorticoid-leptin interaction during development and its impact on developmental programming. In mammals, leptin-induced suppression of glucocorticoid production is critical during development, where leptin prevents stress-induced glucocorticoid production by inducing a period of short-hyporesponsiveness when the adrenal glands fail to respond to certain mild to moderate stressors. Conversely, reduced or absent leptin signaling increases glucocorticoid levels beyond what is appropriate for normal organogenesis. The counterregulatory interactions between leptin and glucocorticoids suggest the potential significant involvement of leptin in disorders that occur from stress during development.

Characterization of a novel corticosterone response gene in Xenopus tropicalis tadpole tails.

Corticosteroids are critical for development and for mediating stress responses across diverse vertebrate taxa. Study of frog metamorphosis has made significant breakthroughs in our understanding of corticosteroid signaling during development in non-mammalian vertebrate species. However, lack of adequate corticosterone (CORT) response genes in tadpoles make identification and quantification of CORT responses challenging. Here, we characterized a CORT-response gene frzb (frizzled related protein) previously identified in Xenopus tropicalis tadpole tail skin by an RNA-seq study. We validated the RNA-seq results that CORT and not thyroid hormone induces frzb in the tails using quantitative PCR. Further, maximum frzb expression was achieved by 100-250 nM CORT within 12-24 hours. frzb is not significantly induced in the liver and brain in response to 100 nM CORT. We also found no change in frzb expression across natural metamorphosis when endogenous CORT levels peak. Surprisingly, frzb is only induced by CORT in X. tropicalis tails and not in Xenopus laevis tails. The exact downstream function of increased frzb expression in tails in response to CORT is not known, but the specificity of hormone response and its high mRNA expression levels in the tail render frzb a useful marker of exogenous CORT-response independent of thyroid hormone for exogenous hormone treatments and in-vivo endocrine disruption studies.

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.

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.

cyp21a2 Knockout Tadpoles Survive Metamorphosis Despite Low Corticosterone.

Corticosteroids are so vital for organ maturation that reduced corticosteroid signaling during postembryonic development causes death in terrestrial vertebrates. Indeed, death occurs at metamorphosis in frogs lacking proopiomelanocortin (pomc) or the glucocorticoid receptor (GR; nr3c1). Some residual corticosteroids exist in pomc mutants to activate the wild-type (WT) GR and mineralocorticoid receptor (MR), and the elevated corticosteroids in GR mutants may activate MR. Thus, we expected a more severe developmental phenotype in tadpoles with inactivation of 21-hydroxylase, which should eliminate all interrenal corticosteroid biosynthesis. Using CRISPR/Cas9 in Xenopus tropicalis, we produced an 11-base pair deletion in cyp21a2, the gene encoding 21-hydroxylase. Growth and development were delayed in cyp21a2 mutant tadpoles, but unlike the other frog models, they survived metamorphosis. Consistent with an absence of 21-hydroxylase, mutant tadpoles had a 95% reduction of aldosterone in tail tissue, but they retained some corticosterone (∼40% of WT siblings), an amount, however, too low for survival in pomc mutants. Decreased corticosteroid signaling was evidenced by reduced expression of corticosteroid-response gene, klf9, and by impaired negative feedback in the hypothalamus-pituitary-interrenal axis with higher messenger RNA expression levels of crh, pomc, star, and cyp11b2 and an approximately 30-fold increase in tail content of progesterone. In vitro tail-tip culture showed that progesterone can transactivate the frog GR. The inadequate activation of GR by corticosterone in cyp21a2 mutants was likely compensated for by sufficient corticosteroid signaling from other GR ligands to allow survival through the developmental transition from aquatic to terrestrial life.

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.

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.

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.

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.

Axial Skeletal Malformations in Genetically Modified Xenopus laevis and Xenopus tropicalis.

Skeletal malformations in captive-bred, adult Xenopus spp., have not previously been reported. Here we describe 10 sexually mature, genetically modified laboratory frogs (6 Xenopus laevis and 4 Xenopus tropicalis) with axial skeletal abnormalities. The young adult frogs were described by veterinary staff as presenting with "hunchbacks," but were otherwise considered to be in good health. All affected frogs were genetically engineered using various techniques: transcription activator-like effector nucleases (TALEN) editing using thyroid hormone receptor α TALEN mRNA, restriction enzyme-mediated integration methods involving insertion of the inducible transgene pCAR/TRDN, or via I-SceI meganuclease transgenesis using either pDRTREdpTR-HS4 or pDPCrtTA-TREG-HS4 plasmid sequences. Radiographic findings (6 frogs) and gross necropsy (10 frogs) revealed vertebral column malformations and sacroiliac deformities that resulted in moderate to severe kyphosis and kyphoscoliosis. These findings were confirmed and additional skeletal abnormalities were identified using computed tomography to create a 3D reconstruction of 4 frogs. Additional findings visible on the 3D reconstructions included incomplete vertebral segmentation, malformed transverse processes, and a short and/or curved urostyle. Histopathologic findings included misshapen intervertebral joints with nonconforming articular surfaces, narrowed joint cavities, flattened or irregularly-formed articular cartilage, irregular maturation lines and nonpolarized chondrocytes, excess fibrocartilage, and evidence of irregular bone resorption and growth. While the specific etiology of the vertebral skeletal abnormalities remains unclear, possibilities include: 1) egg/oocyte physical manipulation (dejellying, microinjection, fertilization, etc.), 2) induction and expression of the transgenes, 3) inactivation (knockout) of existing genes by insertional mutagenesis, or 4) a combination of the above. Furthermore, the possibility of undetected changes in the macro or microenvironment, or a feature of the genetic background of the affected frogs cannot be ruled out.

Corticosterone Is Essential for Survival Through Frog Metamorphosis.

Thyroid hormone (TH) is required for frog metamorphosis, and corticosterone (CORT) increases TH signaling to accelerate metamorphic progression. However, a requirement for CORT in metamorphosis has been difficult to assess prior to the recent development of gene-editing technologies. We addressed this long-standing question using transcription activator-like effector nuclease (TALEN) gene disruption to knock out proopiomelanocortin (pomc) and disrupt CORT production in Xenopus tropicalis. As expected, mutant tadpoles had a reduced peak of plasma CORT at metamorphosis with correspondingly reduced expression of the CORT-response gene Usher syndrome type-1G (ush1g). Mutants had reduced rates of growth and development and exhibited lower expression levels of 2 TH response genes, Krüppel-like factor 9 (klf9) and TH receptor ß (thrb). In response to exogenous TH, mutants had reduced TH response gene induction and slower morphological change. Importantly, death invariably occurred during tail resorption, unless rescued by exogenous CORT and, remarkably, by exogenous TH. The ability of exogenous TH by itself to overcome death in pomc mutants indicates that the CORT-dependent increase in TH signaling may ensure functional organ transformation required for survival through metamorphosis and/or may shorten the nonfeeding metamorphic transition to avoid lethal inanition.

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.

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.

Methods for Investigating the Larval Period and Metamorphosis in Xenopus.

Anuran metamorphosis resembles postembryonic development in mammals, a period around birth when many organs/tissues mature into their adult form as circulating thyroid and stress hormone levels are high. Unlike uterus-enclosed mammalian embryos, tadpoles develop externally and undergo the dramatic changes of hormone-dependent development totally independent of maternal influence, making them a valuable model in which to study vertebrate postembryonic organ development and maturation. Various protocols have been developed and/or adapted for studying metamorphosis in Xenopus laevis and X. tropicalis, two highly related and well-studied frog species. Here, we introduce some of the methods for contemporary cell and molecular studies of gene function and regulation during metamorphosis.

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.

Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas.

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRß, is indispensable for amphibian metamorphosis. However, specific roles for the TRß isoform in metamorphosis are poorly understood. To address this issue, we generated trß-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of trß in metamorphosis. trß-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trß itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of trß Surprisingly, trß-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRß is not required but contributes to the timing of resorptive events of metamorphosis.