Recent advances on the structure and the function relationships of the TRPV4 ion channel.

The members of the superfamily of Transient Receptor Potential (TRP) ion channels are physiologically important molecules that have been studied for many years and are still being intensively researched. Among the vanilloid TRP subfamily, the TRPV4 ion channel is an interesting protein due to its involvement in several essential physiological processes and in the development of various diseases. As in other proteins, changes in its function that lead to the development of pathological states, have been closely associated with modification of its regulation by different molecules, but also by the appearance of mutations which affect the structure and gating of the channel. In the last few years, some structures for the TRPV4 channel have been solved. Due to the importance of this protein in physiology, here we discuss the recent progress in determining the structure of the TRPV4 channel, which has been achieved in three species of animals (Xenopus tropicalis, Mus musculus, and Homo sapiens), highlighting conserved features as well as key differences among them and emphasizing the binding sites for some ligands that play crucial roles in its regulation.

Intestinal remodeling during Xenopus metamorphosis as a model for studying thyroid hormone signaling and adult organogenesis.

Intestinal development takes places in two phases, the initial formation of neonatal (mammals)/larval (anurans) intestine and its subsequent maturation into the adult form. This maturation occurs during postembryonic development when plasma thyroid hormone (T3) level peaks. In anurans such as the highly related Xenopus laevis and Xenopus tropicalis, the larval/tadpole intestine is drastically remodeled from a simple tubular structure to a complex, multi-folded adult organ during T3-dependent metamorphosis. This involved complete degeneration of larval epithelium via programmed cell death and de novo formation of adult epithelium, with concurrent maturation of the muscles and connective tissue. Here, we will summarize our current understanding of the underlying molecular mechanisms, with a focus on more recent genetic and genome-wide studies.

Mechanistic study of transcription factor Sox18 during heart development.

Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.

Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

Identification of tumor-related genes via RNA sequencing of tumor tissues in Xenopus tropicalis.

Cancer treatment is still challenging because the disease is often caused by multiple mutations. Although genomic studies have identified many oncogenes and tumor suppressor genes, gene sets involved in tumorigenesis remain poorly understood. Xenopus, a genus of aquatic frogs, is a useful model to identify gene sets because it can be genetically and experimentally analyzed. Here, we analyzed gene expression in tumor tissues of three individuals in Xenopus tropicalis and identified 55 differentially expressed genes (DEGs). Gene ontology (GO) analysis showed that the upregulated genes in the tumor tissues were enriched in GO terms related to the extracellular matrix and collagen fibril organization. Hierarchical clustering showed that the gene expression patterns of tumor tissues in X. tropicalis were comparable to those of human connective, soft, and subcutaneous tissue-derived cancers. Additionally, pathway analysis revealed that these DEGs were associated with multiple pathways, including the extracellular matrix, collagen fibril organization, MET signaling, and keratan sulfate. We also found that the expression tendency of some DEGs that have not been well analyzed in the cancer field clearly determines the prognosis of human cancer patients. This study provides a remarkable reference for future experimental work on X. tropicalis to identify gene sets involved in human cancer.

Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

Analysis of evolutionary and functional features of the bullfrog SULT1 family.

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.

Expanding EMC foldopathies: Topogenesis deficits alter the neural crest.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.

Cell cycle activation in thyroid hormone-induced apoptosis and stem cell development during Xenopus intestinal metamorphosis.

Amphibian metamorphosis resembles mammalian postembryonic development, a period around birth when many organs mature into their adult forms and when plasma thyroid hormone (T3) concentration peaks. T3 plays a causative role for amphibian metamorphosis. This and its independence from maternal influence make metamorphosis of amphibians, particularly anurans such as pseudo-tetraploid Xenopus laevis and its highly related diploid species Xenopus tropicalis, an excellent model to investigate how T3 regulates adult organ development. Studies on intestinal remodeling, a process that involves degeneration of larval epithelium via apoptosis and de novo formation of adult stem cells followed by their proliferation and differentiation to form the adult epithelium, have revealed important molecular insights on T3 regulation of cell fate during development. Here, we review some evidence suggesting that T3-induced activation of cell cycle program is important for T3-induced larval epithelial cell death and de novo formation of adult intestinal stem cells.

Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.

Cryptochromes (CRYs) are multifunctional molecules that act as a circadian clock oscillating factor, a blue-light sensor, and a light-driven magnetoreceptor. Cry genes are classified into several groups based on the evolutionary relationships. Cryptochrome 6 gene (Cry6) is present in invertebrates and lower vertebrates such as amphibians and fishes. Here we identified a Cry6 ortholog in Xenopus tropicalis (XtCry6). XtCRY6 retains a conserved long N-terminal extension (termed CRY N-terminal extension; CNE) that is not found in any CRY in the other groups. A structural prediction suggested that CNE contained unique structures; a tetrahelical fold structure topologically related to KaiA/RbsU domain, overlapping nuclear- and nucleolar-localizing signals (NLS/NoLS), and a novel motif (termed DI-UIM) overlapping a double-sided ubiquitin-interacting motif (DUIM) and an inverted ubiquitin-interacting motif (IUIM). Potential activities of the NLS/NoLS and DI-UIM were examined to infer the molecular function of XtCRY6. GFP-NLS/NoLS fusion protein exogenously expressed in HEK293 cells was mostly observed in the nucleolus, while GFP-XtCRY6 was observed in the cytoplasm. A glutathione S-transferase (GST) pull-down assay suggested that the DI-UIM physically interacts with polyubiquitin. Consistently, protein docking simulations implied that XtCRY6 DI-UIM binds two ubiquitin molecules in a relationship of a twofold rotational symmetry with the symmetry axis parallel or perpendicular to the DI-UIM helix. These results strongly suggested that XtCRY6 does not function as a circadian transcriptional repressor and that it might have another function such as photoreceptive molecule regulating light-dependent protein degradation or gene expression through a CNE-mediated interaction with ubiquitinated proteins in the cytoplasm and/or nucleolus.

Activation of P53 pathway contributes to Xenopus hybrid inviability.

Hybrid incompatibility as a kind of reproductive isolation contributes to speciation. The nucleocytoplasmic incompatibility between Xenopus tropicalis eggs and Xenopus laevis sperm (te×ls) leads to specific loss of paternal chromosomes 3L and 4L. The hybrids die before gastrulation, of which the lethal causes remain largely unclear. Here, we show that the activation of the tumor suppressor protein P53 at late blastula stage contributes to this early lethality. We find that in stage 9 embryos, P53-binding motif is the most enriched one in the up-regulated Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) peaks between te×ls and wild-type X. tropicalis controls, which correlates with an abrupt stabilization of P53 protein in te×ls hybrids at stage 9. Inhibition of P53 activity via either tp53 knockout or overexpression of a dominant-negative P53 mutant or Murine double minute 2 proto-oncogene (Mdm2), a negative regulator of P53, by mRNA injection can rescue the te×ls early lethality. Our results suggest a causal function of P53 on hybrid lethality prior to gastrulation.

Evolutionary and functional conservation of myeloid differentiation factor 88 (MyD88) in amphibian Xenopus tropicalis.

As a universal adaptor used by most TLR members, the myeloid differentiation factor 88 (MyD88) plays essential roles in TLR-mediated inflammatory response of invertebrate and vertebrate animals, and functional features of MyD88 remain largely unknown in amphibians. In this study, a MyD88 gene named Xt-MyD88 was characterized in the Western clawed frog (Xenopus tropicalis). Xt-MyD88 and MyD88 in other species of vertebrates share similar structural characteristics, genomic structures, and flanking genes, suggesting that MyD88 is structurally conserved in different phyla of vertebrates ranging from fish to mammals. Moreover, Xt-MyD88 was widely expressed in different organs/tissues, and was induced by poly(I:C) in spleen, kidney, and liver. Importantly, overexpression of Xt-MyD88 triggered a marked activation of both NF-κB promoter and interferon-stimulated response elements (ISREs), implying that it may be play important roles in inflammatory responses of amphibians. The research represents the first characterization on the immune functions of amphibian MyD88, and reveals considerable functional conservation of MyD88 in early tetrapods.

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.

Identification and functional characterization of protein kinase R (PKR) in amphibian Xenopus tropicalis.

As one of interferon-induced serine/threonine kinases, the protein kinase R (PKR) plays vital roles in antiviral defense, and functional features of PKR remain largely unknown in amphibians, which suffer from ranaviral diseases in the last few decades. In this study, a PKR gene named Xt-PKR was characterized in the Western clawed frog (Xenopus tropicalis). Xt-PKR gene was widely expressed in different organs/tissues, and was rapidly induced by poly(I:C) in spleen, kidney, and liver. Intriguingly, Xt-PKR could be up-rugulated by the treatment of type I and type III interferons, and the transcript level of Xt-PKR induced by type I interferon was much higher than that of type III interferon. Moreover, overexpression of Xt-PKR can suppress the protein synthesis and ranavirus replication in vitro, and the residue lysine required for the translation inhibition activity in mammalian PKR is conserved in Xt-PKR. The present study represents the first characterization on the functions of amphibian PKR, and reveals considerable functional conservation of PKR in early tetrapods.

Comparative Analysis of Transcriptome Profiles Reveals Distinct and Organ-Dependent Genomic and Nongenomic Actions of Thyroid Hormone in Xenopus tropicalis Tadpoles.

Background: Thyroid hormone (triiodothyronine [T3]) is essential for development and organ metabolism in all vertebrates. T3 has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via T3 receptors (TRs) in vertebrate development, mostly through TR-knockout and TR-knockin studies, little is known about the effects of T3 on gene expression in animals in the absence of TR. We have been studying Xenopus metamorphosis as a model for mammalian postembryonic development, a period around birth when plasma T3 level peaks and many organs/tissues mature into their adult forms. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals. This offers an opportunity to compare the effects of T3 on global gene expression in tadpole tissues in the presence or absence of TR. Methods: We analyzed the effects of T3 on gene expression in tadpole tail and intestine by using RNA-seq analysis on wild-type and TRDKO tadpoles with or without T3 treatment. Results: We observed that removing TRs reduced the number of genes regulated by T3 in both organs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that T3 affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms and KEGG pathways that were enriched among genes regulated in wild-type tissues are likely involved in mediating the effects of T3 on metamorphosis, for example, those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among T3-regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to "metabolism" and "immune response" were highly enriched among T3-regulated genes. We further observed strong divergence in the TR-independent nongenomic effects of T3 in the intestine and tail. Conclusions: Our data suggest that T3 has distinct and organ-dependent effects on gene expression in developing tadpoles. The TR-mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of T3 on metamorphosis. T3 appears to have a major effect on metabolism and immune response via TR-independent nongenomic processes.

Molecular cloning and analysis of the ghrelin/GHSR system in Xenopus tropicalis.

Ghrelin is a gut-derived peptide with several physiological functions, including feeding, gastrointestinal motility, and hormonal secretion. Recently, a host defense peptide, liver-expressed antimicrobial peptide-2 (LEAP2), was reported as an endogenous antagonist of growth hormone secretagogue receptor (GHS-R). The physiological relevance of the molecular LEAP2-GHS-R interaction in mammals has been explored; however, studies on non-mammals are limited. Here, we report the identification and functional characterization of ghrelin and its related molecules in Western clawed frog (Xenopus tropicalis), a known model organism. We first identified cDNA encoding X. tropicalis ghrelin and GHS-R. RT-qPCR revealed that ghrelin mRNA expression was most abundant in the stomach. GHS-R mRNA was widely distributed in the brain and peripheral tissues, and a relatively strong signal was observed in the stomach and intestine. In addition, LEAP2 was mainly expressed in intestinal tissues at higher levels than in the liver. In functional analysis, X. tropicalis ghrelin and human ghrelin induced intracellular Ca2+ mobilization with EC50 values in the low nanomolar range in CHO-K1 cells expressing X. tropicalis GHS-R. Furthermore, ghrelin-induced GHS-R activation was antagonized with IC50 values in the nanomolar range by heterologous human LEAP2. We also validated the expression of ghrelin and feeding-related factors under fasting conditions. After 2 days of fasting, no changes in ghrelin mRNA levels were observed in the stomach, but GHS-R mRNA levels were significantly increased, associated with significant downregulation of nucb2. In addition, LEAP2 upregulation was observed in the duodenum. These results provide the first evidence that LEAP2 functions as an antagonist of GHS-R in the anuran amphibian X. tropicalis. It has also been suggested that the ghrelin/GHS-R/LEAP2 system may be involved in energy homeostasis in X. tropicalis.

Organ-specific effects on target binding due to knockout of thyroid hormone receptor α during Xenopus metamorphosis.

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRß. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thratmshi ) tadpoles, we have previously shown that TRα is important for T3-dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild-type and Xtr·thratmshi tails with or without T3 treatment by using chromatin immunoprecipitation-sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild-type but not Xtr·thratmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating T3-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.

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.

Human Melanocortin-2 Receptor: Identifying a Role for Residues in the TM4, EC2, and TM5 Domains in Activation and Trafficking as a Result of Co-Expression with the Accessory Protein, Mrap1 in Chinese Hamster Ovary Cells.

Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.

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.