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Even though Sugars Will Eventually be Exported Transporters (SWEETs) have been found in every sequenced plant genome, a comprehensive understanding of their functionality is lacking. In this study, we focused on the SWEET family of barley (Hordeum vulgare). A radiotracer assay revealed that expressing HvSWEET11b in African clawed frog (Xenopus laevis) oocytes facilitated the bidirectional transfer of not only just sucrose and glucose, but also cytokinin. Barley plants harboring a loss-of-function mutation of HvSWEET11b could not set viable grains, while the distribution of sucrose and cytokinin was altered in developing grains of plants in which the gene was knocked down. Sucrose allocation within transgenic grains was disrupted, which is consistent with the changes to the cytokinin gradient across grains, as visualized by magnetic resonance imaging and Fourier transform infrared spectroscopy microimaging. Decreasing HvSWEET11b expression in developing grains reduced overall grain size, sink strength, the number of endopolyploid endosperm cells, and the contents of starch and protein. The control exerted by HvSWEET11b over sugars and cytokinins likely predetermines their synergy, resulting in adjustments to the grain's biochemistry and transcriptome. [ABSTRACT FROM AUTHOR]

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and the third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers, and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function. [ABSTRACT FROM AUTHOR]

The normal stages of embryonic development for wild‐type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high‐quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild‐type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians. [ABSTRACT FROM AUTHOR]

Idiopathic hypocalcemia in Thoroughbred (TB) foals causes tetany and seizures and is invariably fatal. Based upon the similarity of this disease with human familial hypoparathyroidism and occurrence only in the TB breed, we conducted a genetic investigation on two affected TB foals. Familial hypoparathyroidism was identified, and pedigree analysis suggested an autosomal recessive (AR) mode of inheritance. We performed whole-genome sequencing of the two foals, their unaffected dams and four unaffected, unrelated TB horses. Both homozygosity mapping and an association analysis were used to prioritize potential genetic variants. Of the 2,808 variants that significantly associated with the phenotype using an AR mode of inheritance (PA,p.Ser875*) was significantly associated with the hypoparathyroid phenotype (Pallelic = 0.008). Affected foals were homozygous for the variant, with two additional affected foals subsequently confirmed in 2019. Necropsies of all affected foals failed to identify any histologically normal parathyroid glands. Because the nonsense mutation in RAPGEF5 was near the C-terminal end of the protein, the impact on protein function was unclear. Therefore, we tested the variant in our Xenopus overexpression model and demonstrated RAPGEF5 loss-of-function. This RAPGEF5 variant represents the first genetic variant for hypoparathyroidism identified in any domestic animal species.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

Animal development and homeostasis depend on precise temporal and spatial intercellular signaling. Components shared between signaling pathways, generally thought to decrease specificity, paradoxically can also provide a solution to pathway coordination. Here we show that the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways share Apcdd1 as a common inhibitor and that Apcdd1 is a taxon-restricted gene with novel domains and signaling functions. Previously, we showed that Apcdd1 inhibits Wnt signaling (Shimomura et al., 2010), here we find that Apcdd1 potently inhibits BMP signaling in body axis formation and neural differentiation in chicken, frog, zebrafish. Furthermore, we find that Apcdd1 has an evolutionarily novel protein domain. Our results from experiments and modeling suggest that Apcdd1 may coordinate the outputs of two signaling pathways that are central to animal development and human disease.

Size is a fundamental feature of biology that affects physiology at all levels, from the organism to organs and tissues to cells and subcellular structures. How size is determined at these different levels, and how biological structures scale to fit together and function properly are important open questions. Historically, amphibian systems have been extremely valuable to describe scaling phenomena, as they occupy some of the extremes in biological size and are amenable to manipulations that alter genome and cell size. More recently, the application of biochemical, biophysical, and embryological techniques to amphibians has provided insight into the molecular mechanisms underlying scaling of subcellular structures to cell size, as well as how perturbation of normal size scaling impacts other aspects of cell and organism physiology.

Cellular Ca2+ signals are often constrained to cytosolic micro- or nano-domains where stochastic openings of Ca2+ channels cause large fluctuations in local Ca2+ concentration (Ca2+ 'noise'). With the advent of TIRF microscopy to image the fluorescence of Ca2+-sensitive probes from attoliter volumes it has become possible to directly monitor these signals, which closely track the gating of plasmalemmal and ER Ca2+-permeable channels. Nevertheless, it is likely that many physiologically important Ca2+ signals are too small to resolve as discrete events in fluorescence recordings. By analogy with noise analysis of electrophysiological data, we explore here the use of statistical approaches to detect and analyze such Ca2+ noise in images obtained using Ca2+-sensitive indicator dyes. We describe two techniques - power spectrum analysis and spatio-temporal correlation - and demonstrate that both effectively identify discrete, spatially localized calcium release events (Ca2+ puffs). Moreover, we show they are able to detect localized noise fluctuations in a case where discrete events cannot directly be resolved.

Synaptic communication requires the expression of functional postsynaptic receptors that match the presynaptically released neurotransmitter. The ability of neurons to switch the transmitter they release is increasingly well documented, and these switches require changes in the postsynaptic receptor population. Although the activity-dependent molecular mechanism of neurotransmitter switching is increasingly well understood, the basis of specification of postsynaptic neurotransmitter receptors matching the newly expressed transmitter is unknown. Using a functional assay, we show that sustained application of glutamate to embryonic vertebrate skeletal muscle cells cultured before innervation is necessary and sufficient to up-regulate ionotropic glutamate receptors from a pool of different receptors expressed at low levels. Up-regulation of these ionotropic receptors is independent of signaling by metabotropic glutamate receptors. Both imaging of glutamate-induced calcium elevations and Western blots reveal ionotropic glutamate receptor expression prior to immunocytochemical detection. Sustained application of glutamate to skeletal myotomes in vivo is necessary and sufficient for up-regulation of membrane expression of the GluN1 NMDA receptor subunit. Pharmacological antagonists and morpholinos implicate p38 and Jun kinases and MEF2C in the signal cascade leading to ionotropic glutamate receptor expression. The results suggest a mechanism by which neuronal release of transmitter up-regulates postsynaptic expression of appropriate transmitter receptors following neurotransmitter switching and may contribute to the proper expression of receptors at the time of initial innervation.

For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.

The structure of pannexin 1, a channel protein with a large pore, has been determined for the first time.

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 pseudotetraploid 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Somatic and germline gain‐of‐function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor‐prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss‐of‐function of the proto‐oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure‐based prediction and functional tests using human knock‐in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress‐induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis. Synopsis: A novel recessive variant in RAF1 was identified as a possible cause of a hitherto unknown lethal neonatal syndrome with progeroid features. This private p.Thr543Met mutation abolished downstream ERK pathway signaling while increasing susceptibility to apoptosis via ASK1 de‐repression. A homozygous p.Thr543Met mutation in RAF1 was found in a neonate presenting with a syndrome encompassing cutaneous, craniofacial, cardiac and limb anomalies.In cultured cells and in vivo using Xenopus assays the p.Thr543Met mutation behaved as a loss‐of‐function allele that abolishes downstream ERK pathway signaling in response to growth factor stimulation.p.Thr543Met knock‐in in 293T cells revealed that RAF1T543M is unstable and more sensitive to stress‐induced apoptosis via ASK1 de‐repression.These findings describe, for the first time, the consequences of a loss of RAF1 function during human development, contrasting with gain‐of‐function mutations seen in cancer‐prone RASopathies. [ABSTRACT FROM AUTHOR]

Direct discharge of aquaculture wastewater may have toxic effects, due to the presence of heavy metals, antibiotics, and even resistant pathogens, but little attention has been given. Here, tanks simulating a wild ecosystem were built to study the effects of long-term exposure to duck wastewater containing oxytetracycline (OTC) and/or arsenic (As) on the growth, physiological function, and gut microbiota evolution of Xenopus tropicalis. The results showed that duck wastewater had no apparent impact on X. tropicalis , but the impact increased significantly (P < 0.05) with exposure to OTC and/or As, especially the impact on body weight and growth rate. Biochemical indicators revealed varying degrees of oxidative stress damage, hepatotoxicity (inflammation, necrosis, and sinusoids), and collagen fibrosis of X. tropicalis in all treated groups after 72 days of exposure, which indirectly inhibited X. tropicalis growth. Moreover, 16S rDNA amplicon sequencing results showed that the gut microbiota structure and metabolic function were perturbed after chronic exposure, which might be the leading cause of growth inhibition. Interestingly, the abundance of intestinal resistance genes (RGs) increased with exposure time owing to the combined direct and indirect effects of stress factors in duck wastewater. Moreover, once the RGs were expressed, the resistance persisted for at least 24 days, especially that conferred by tet A. These results provide evidence of the toxic effects of DW containing OTC (0.1–4.0 mg/L) and/or As (0.3–3.5 µg/L) on amphibians and indicate that it is vital to limit the usage of heavy metals and antibiotics on farms to control the biotoxicity of wastewater. [Display omitted] [ABSTRACT FROM AUTHOR]

Xenbase (https://www.xenbase.org/), the Xenopus model organism knowledgebase, is a web-accessible resource that integrates the diverse genomic and biological data from research on the laboratory frogs Xenopus laevis and Xenopus tropicalis. The goal of Xenbase is to accelerate discovery and empower Xenopus research, to enhance the impact of Xenopus research data, and to facilitate the dissemination of these data. Xenbase also enhances the value of Xenopus data through high-quality curation, data integration, providing bioinformatics tools optimized for Xenopus experiments, and linking Xenopus data to human data, and other model organisms. Xenbase also plays an indispensable role in making Xenopus data interoperable and accessible to the broader biomedical community in accordance with FAIR principles. Xenbase provides annotated data updates to organizations such as NCBI, UniProtKB, Ensembl, the Gene Ontology consortium, and most recently, the Alliance of Genomic Resources, a common clearing house for data from humans and model organisms. This article provides a brief overview of key and recently added features of Xenbase. New features include processing of Xenopus high-throughput sequencing data from the NCBI Gene Expression Omnibus; curation of anatomical, physiological, and expression phenotypes with the newly created Xenopus Phenotype Ontology; Xenopus Gene Ontology annotations; new anatomical drawings of the Normal Table of Xenopus development; and integration of the latest Xenopus laevis v10.1 genome annotations. Finally, we highlight areas for future development at Xenbase as we continue to support the Xenopus research community. [ABSTRACT FROM AUTHOR]

The abrupt morphological changes of the intestine during metamorphosis have been detailed in frogs. The features of intestinal metamorphosis are shortening of the intestine and remodeling of the intestinal epithelium. It is believed that the purpose of the morphological changes of the intestine is adaptation from aquatic herbivorous to carnivorous life. However, little is known about the physiological importance of these morphological changes. To elucidate the functional changes during metamorphosis, we measured luminal Na+ concentrations and Na+-dependent glucose uptake in tadpoles and adult African clawed frogs Xenopus laevis. The small intestine was isolated and divided into four segments in length, the luminal contents collected for analysis of ion concentration by ion chromatography. Phlorizin-sensitive glucose-induced short-circuit current (DIsc) was measured in intestinal preparations mounted in Ussing chambers. Although dietary sodium intake was extremely low in tadpoles, luminal Na+ concentration gradually increased along the proximal to the middle part of the intestine (>70 mM), and this Na+ concentration was comparable with that of carnivorous adult frogs. The increment of glucose-induced DIsc was observed in tadpole intestine. We also measured the DIsc induced by acetic acid, which is the major short-chain fatty acid produced by fermentation. The expression levels of mRNA for Na+-dependent glucose transporter 1 and tight junction protein claudin-15 in each intestinal segment was measured. These results suggest that luminal Na+ homeostasis is important and luminal Na+ is kept at a high concentration for Na+-dependent nutrient absorption mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine–pyrimidine (6–4) photoproducts (6–4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6–4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light‐exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies. [ABSTRACT FROM AUTHOR]

The biological function of the novel zinc-finger SWIM domain-containing protein family (ZSWIM) during embryonic development remains elusive. Here, we conducted a genome-wide analysis to explore the evolutionary processes of the ZSWIM gene family members in mice, Xenopus tropicalis, zebrafish, and humans. We identified nine putative ZSWIM genes in the human and mouse genome, eight in the Xenopus genome, and five in the zebrafish genome. Based on multiple sequence alignment, three members, ZSWIM5, ZSWIM6, and ZSWIM8, demonstrated the highest homology across all four species. Using available RNA sequencing (RNAseq) data, ZSWIM genes were found to be widely expressed across different tissues, with distinct tissuespecific properties. To identify the functions of the ZSWIM protein family during embryogenesis, we examined temporal and spatial expression patterns of zswim family genes in Xenopus embryos. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that each member had a distinct expression profile. Whole-mount in situ hybridization showed that both zswim1 and zswim3 were maternally expressed genes; zswim5 and zswim6 were expressed throughout embryogenesis and displayed dynamic expression in the brain, eyes, somite, and bronchial arch at the late tailbud stages; zswim7 was detected in the eye area; zswim8 showed a dynamic expression pattern during the tailbud stages, with expression detected in the brain, eyes, and somite; zswim9 was faintly expressed throughout embryonic development. This study provides a foundation for future research to delineate the functions of ZSWIM gene members. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (FGFs) encode a large family of growth factor proteins that activate several intracellular signaling pathways to control diverse physiological functions. The human genome encodes 22 FGFs that share a high sequence and structural homology with those of other vertebrates. FGFs orchestrate diverse biological functions by regulating cellular differentiation, proliferation, and migration. Dysregulated FGF signaling may contribute to several pathological conditions, including cancer. Notably, FGFs exhibit wide functional diversity among different vertebrates spatiotemporally. A comparative study of FGF receptor ligands and their diverse roles in vertebrates ranging from embryonic development to pathological conditions may expand our understanding of FGF. Moreover, targeting diverse FGF signals requires knowledge regarding their structural and functional heterogeneity among vertebrates. This study summarizes the current understanding of human FGF signals and correlates them with those in mouse and Xenopus models, thereby facilitating the identification of therapeutic targets for various human disorders. [ABSTRACT FROM AUTHOR]

Comparative and ecological aspects of the reorganizations of early development in the class Amphibia are analyzed. We used data on the developmental diversity in a number of families belonging to the orders Anura and Caudata, in which many species had lost their connection with the aquatic environment. Model representatives of the class Amphibia (Ambystoma mexicanum, Rana temporaria, and Xenopus laevis) have small eggs (no more than 2.5 mm in diameter). In these species, the slowdown in the rate of cell divisions and the loss of synchrony occur at the midblastula stage. However, phylogenetically basal amphibian species (Ascaphus truei, Cryptobranchus alleganiensis) are characterized by the large (4–6 mm in diameter) yolky eggs and a short series of synchronous blastomere divisions (the synchrony is already lost at the 8-cell stage of cleavage). They do not have a "midblastula transition," which is characteristic of the above model species. On the other hand, many evolutionarily advanced non-model species of caudate and anuran amphibians (for example, Desmognathus fuscus, Gastrotheca riobambae, Philoria sphagnicolus), as well as the basal species, are characterized by the large, yolk-rich eggs and the early loss of cell division synchrony. Phylogenetic analysis suggests that the cleavage pattern of the most extensively studied amphibians, the Mexican axolotl (Caudata) and the African clawed frog (Anura), represents a homoplasy. The midblastula transition, which is characteristic of these two species, might have evolved convergently in these two orders of amphibians as an embryonic adaptation to development in lentic water. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The nervous system has various types of cells derived from three neuroectodermal regions: neural plate (NP), neural crest (NC), and preplacodal ectoderm (PPE). Differentiation of these regions is regulated by various morphogens. However, regulatory mechanisms of morphogen distribution in neural patterning are still debated. In general, an extracellular component, heparan sulfate (HS), is essential to regulate morphogen gradients by modulating morphogen binding. The present study focused on an HS modification enzyme, heparan sulfate 6-O-sulfotransferase 1 (Hs6st1), which is highly expressed during the neurula stage in Xenopus. Our present in situ hybridization analysis revealed that Hs6st1 is expressed in the lateral sensorial layer of neuroectoderm. Overexpression of Hs6st1 expands Sox3 (NP marker gene) expression, and slightly dampens FoxD3 (NC marker) expression. Hs6st1 knockout using the CRISPR/Cas9 system also expands the neural plate region, followed by retinal malformation. These results imply that 6-O sulfation, mediated by Hs6st1, selectively regulates morphogen distribution required for neuroectodermal patterning. Among morphogens required for patterning, Fgf8a accumulates on Hs6st1 -expressing cells, whereas a secreted BMP antagonist, Noggin, diffuses away from those cells. Thus, cell-autonomous 6-O sulfation of HS at the sensorial layer of neuroectoderm also affects neuroectodermal patterning in neighboring regions, including neural plate and neural crest, not only through accumulation, but also through dispersal of specific morphogens. [Display omitted] • Hs6st1 is expressed in lateral sensorial layer of neuroectoderm in Xenopus neurula. • Hs6st1 organizes neuroectodermal patterning and subsequent retinal formation. • 6-O-sulfation of heparan sulfate mediated by Hs6st1 accumulates Fgf8a but not Noggin. [ABSTRACT FROM AUTHOR]

We characterized Xenopus laevis C‐C motif chemokine ligand 19.L (ccl19.L) and C‐C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L‐overexpressing explants attracted cells at a distance and ccl21.L‐overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis‐like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal–ventral patterning during early embryogenesis in Xenopus. [ABSTRACT FROM AUTHOR]

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen‐mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N‐deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked‐down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N‐sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1‐expressing cells, but that it inhibits signaling in non‐ndst1‐expressing cells, leading to proper neuroectodermal patterning. [ABSTRACT FROM AUTHOR]

Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter. [ABSTRACT FROM AUTHOR]

Many diseases of Xenopus laevis frogs have been described in the literature however, it is often difficult to find images which clearly illustrate associated clinical signs. This poster has been designed as a refinement to bridge this gap using photographs taken of frogs with clinical signs. My aims are: - To share my experiences, via images, to help improve the welfare and husbandry of this species. - To stimulate a dialogue which results in further sharing of ideas and information. [ABSTRACT FROM AUTHOR]

There is a wide variety of different types of mouse enrichment currently available, some items of which are more practical than others. Validating items used for providing enrichment can be time consuming and costly, nevertheless it is important to ensure that any objects being provided for this purpose are truly offering enrichment opportunities for the animals. The purpose of this study was to demonstrate how a quick and simple cage side assessment can be undertaken to identify which items are most effective as enrichments and whether they offer short- or long-term solutions. The study compared four different types of enrichment, all of which are easily available, in five cages of pair housed 6-monthold CD1 mice. Through this approach, we found that the mice preferred items suspended from the cage lid. Using this information, we have identified a practical and cost-effective solution and confirmed that it maintains novelty over longer periods. This validated enrichment is now being used routinely in the Biomedical Services Unit at the University of Birmingham. [ABSTRACT FROM AUTHOR]

Digital photography has been used at The Gurdon Institute, Cambridge University since 2016, primarily as an identification aid. We have found it to be: non-invasive, quick, cheap, reliable and accessible. We also discovered it to be advantageous in monitoring the health and body condition of our large colony of over 600 Xenopus laevis frogs. This has been an excellent refinement in their welfare and care. [ABSTRACT FROM AUTHOR]

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Marine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin‐like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts. [ABSTRACT FROM AUTHOR]

The reciprocal inhibition between two signaling centers, the Spemann organizer (dorsal mesoderm) and ventral region (mesoderm and ectoderm), collectively regulate the overall development of vertebrate embryos. Each center expresses key homeobox transcription factors (TFs) that directly control target gene transcription. Goosecoid (Gsc) is an organizer (dorsal mesoderm)-specific TF known to induce dorsal fate and inhibit ventral/ectodermal specification. Ventx1.1 (downstream of Bmp signaling) induces the epidermal lineage and inhibits dorsal organizer-specific genes from the ventral region. Chordin (Chrd) is an organizer-specific secreted Bmp antagonist whose expression is primarily activated by Gsc. Alternatively, chrd expression is repressed by Bmp/Ventx1.1 in the ventral/epidermal region. However, the regulatory mechanisms underlying the transcription mediated by Gsc and Ventx1.1 remain elusive. Here, we found that the chrd promoter contained two cis-acting response elements that responded negatively to Ventx1.1 and positively to Gsc. In the ventral/ectodermal region, Ventx1.1 was directly bound to the Ventx1.1 response element (VRE) and inhibited chrd transcription. In the organizer region, Gsc was bound to the Gsc response elements (GRE) to activate chrd transcription. The Gsc-mediated positive response on the chrd promoter completely depended on another adjacent Wnt response cis-acting element (WRE), which was the TCF7 (also known as Tcf1) binding element. Site-directed mutagenesis of VRE, GRE, or WRE completely abolished the repressive or activator activity of Ventx1.1 and Gsc, respectively. The ChIP-PCR results confirmed the direct binding of Ventx1.1 and Gsc/Tcf7 to VRE and GRE/WRE, respectively. These results demonstrated that chrd expression is oppositely modulated by homeobox TFs, Ventx1.1, and Gsc/Tcf7 during the embryonic patterning of Xenopus gastrula. [ABSTRACT FROM AUTHOR]

Ex situ amphibian populations are a key component of global amphibian conservation strategy, and optimal husbandry is vital to ex situ conservation success. Animal behavior can be used to inform captive welfare and improve husbandry practices. However, it has been little used for amphibians compared with mammals and birds. The goal of this study was to explore the effect of different colored tank backgrounds on the behavior of the critically endangered Lake Oku clawed frog (Xenopus longipes) in captivity. This was conducted by studying the behavior of a group of 24 captive frogs in 5 groups using established behavioral indicators of presumed stress. Resting and swimming behaviors, established in the literature as linked to acute stress, were recorded under conditions of three background colors and a standard husbandry control of no background. Frog groups were exposed to each background for five days with behavioral frequencies recorded daily from 11:00 until 13:00. Using generalized linear mixed models, we identified a significant effect of background days after the background was changed and the interaction between the two variables on both swimming and resting behavior. The results of this study suggest an initial response of stress to altering the background, modulated by the color of the background, followed by the extinction of the stress response such that by five days after the background change, behaviors were similar to the baseline and indistinguishable between treatments. Overall, this study suggests that frog stress behavior was not differentially directly affected by green, grey, black, or transparent backgrounds but that green and grey backgrounds were associated with the smallest stress response to background change. These colors may therefore be recommended to reduce the impact of stress from disturbance. [ABSTRACT FROM AUTHOR]

The African clawed frog, Xenopus laevis, has been reported to tolerate long-term fasting without dormancy. However, the strategies for energy acquisition during fasting are unclear in this species. We performed 3- and 7-month fasting experiments to investigate how the metabolism of male X. laevis changes during long-term fasting. We found that the levels of several serum biochemical parameters, such as glucose, triglycerides, and free fatty acids, as well as liver glycogen were reduced after 3 months of fasting, whereas after 7 months of fasting, triglyceride levels were reduced, and fat body wet weight was lower than that of fed group indicating the onset of lipid catabolism. In addition, transcript levels of gluconeogenic genes, such as pck1, pck2, g6pc1.1, and g6pc1.2, were increased in the livers of animals fasted for 3 months, suggesting upregulation of gluconeogenesis. Our results raise the possibility that male X. laevis can tolerate much longer fasting than previously reported by utilizing several energy storage molecules. Further investigation of the effects of prolonged fasting on the metabolic switches from carbohydrates to lipids or amino acids in X. laevis is required. [ABSTRACT FROM AUTHOR]

Congenital Heart Disease (CHD) is the most common birth defect and leading cause of infant mortality, yet molecular mechanisms explaining CHD remain mostly unknown. Sequencing studies are identifying CHD candidate genes at a brisk rate including MINK1 , a serine/threonine kinase. However, a plausible molecular mechanism connecting CHD and MINK1 is unknown. Here, we reveal that mink1 is required for proper heart development due to its role in left-right patterning. Mink1 regulates canonical Wnt signaling to define the cell fates of the Spemann Organizer and the Left-Right Organizer, a ciliated structure that breaks bilateral symmetry in the vertebrate embryo. To identify Mink1 targets, we applied an unbiased proteomics approach and identified the high mobility group architectural transcription factor, Hmga2. We report that Hmga2 is necessary and sufficient for regulating Spemann's Organizer. Indeed, we demonstrate that Hmga2 can induce Spemann Organizer cell fates even when β-catenin, a critical effector of the Wnt signaling pathway, is depleted. In summary, we discover a transcription factor, Hmga2, downstream of Mink1 that is critical for the regulation of Spemann's Organizer, as well as the LRO, defining a plausible mechanism for CHD. [Display omitted] • MINK1 is a novel regulator of left-right patterning and heart development. • MINK1 regulates canonical Wnt signaling during induction of the Spemann Organizer. • HMGA2 is an indirect target of MINK1 phosphorylation. • HMGA2 functions downstream of b-catenin during induction of Spemann Organizer. [ABSTRACT FROM AUTHOR]

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ selfinhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation. [ABSTRACT FROM AUTHOR]

Solute carrier 12 (Slc12) is a family of electroneutral cation-coupled chloride (Cl−) cotransporters. Na+/K+/2Cl− (Nkcc) and Na+/Cl− cotransporters (Ncc) belong to the Nkcc/Ncc subfamily. Human and mouse possess one gene for the Na+/Cl− cotransporter (ncc gene: slc12a3), whereas teleost fishes possess multiple ncc genes, slc12a3 (ncc1) and slc12a10 (ncc2), in addition to their species-specific paralogs. Amphibians and squamates have two ncc genes: slc12a3 (ncc1) and ncc3. However, the evolutionary relationship between slc12a10 and ncc3 remains unresolved, and the presence of slc12a10 (ncc2) in mammals has not been clarified. Synteny and phylogenetic analyses of vertebrate genome databases showed that ncc3 is the ortholog of slc12a10, and slc12a10 is present in most ray-finned fishes, coelacanths, amphibians, reptiles, and a few mammals (e.g., platypus and horse) but pseudogenized or deleted in birds, most mammals, and some ray-finned fishes (pufferfishes). This shows that slc12a10 is widely present among bony vertebrates and pseudogenized or deleted independently in multiple lineages. Notably, as compared with some fish that show varied slc12a10 tissue expression profile, spotted gar, African clawed frog, red-eared slider turtle, and horse express slc12a10 in the ovaries or premature gonads. In horse tissues, an unexpectedly large number of splicing variants for Slc12a10 have been cloned, many of which encode truncated forms of Slc12a10, suggesting that the functional constraints of horse slc12a10 are weakened, which may be in the process of becoming a pseudogene. Our results elaborate on the evolution of Nkcc/Ncc subfamily of Slc12 in vertebrates. [ABSTRACT FROM AUTHOR]

Background: Xenopus frogs are used extensively for modeling genetic diseases owing to characteristics such as the abundance of eggs combined with their large size, allowing easy manipulation, and rapid external embryo development enabling the examination of cellular and phenotypic alterations throughout embryogenesis. However, genotyping of mutant animals is currently done either as part of a large group, requiring many embryos, or late in development with welfare effects. Therefore, we adapted the Zebrafish Embryonic Genotyper for rapid genomic DNA extraction from Xenopus tropicalis and Xenopus laevis at early stages. Results: Sufficient and good quality DNA was extracted as early as the Nieuwkoop and Faber stage 19 and, importantly, no detrimental effects of the extraction process on the subsequent tadpole development, behavior, or morphology were observed. Amplicons of up to 800 bp were successfully amplified and used for further analyses such as gel electrophoresis, T7 endonuclease I assay and Sanger sequencing. Conclusion: This method allows rapid genotyping during the early stages of Xenopus development, which enables safe identification of mutants. These can be analyzed at early developmental stages or selected for raising without the need for invasive genotyping later, with resource savings and ethical gains in line with the 3Rs principles. Key Findings: Xenopus frogs are used extensively for modelling genetic diseases. However, current procedures for genotyping of mutant animals require many embryos, or are done late in development, which hampers experiments have welfare effects. Therefore, we developed a method allowing rapid genotyping during the early stages of Xenopus development, which has no detrimental effects on the subsequent tadpole growth, with resource savings and ethical gains in line with the 3Rs principles. [ABSTRACT FROM AUTHOR]

The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. The primary endpoints included mortality, developmental stage, embryo‐larval malformation, behavioral effects, hindlimb length (HLL), growth (snout‐vent length [SVL] and wet body weight), and histopathology. The 28‐day LC50 value with 95% confidence intervals was 61.2 (51.4–72.9) μg Cu/L with 250 μg Cu/L resulting in complete lethality. Developmental arrest in the 82.5 and delay in the 27.2 μg Cu/L treatments was observed as early as study day 10 continuing throughout the remainder of exposure. SVL‐normalized HLL, body weight, and SVL in the 27.2 and 82.5 μg Cu/L treatments were significantly decreased relative to control. At 82.5 μg Cu/L, and thyroid gland size was markedly reduced when compared with controls consistent with the stage of developmental and growth arrest. Concentration‐dependent findings in the intestine, liver, gills, eyes, and pharyngeal mucosa were consistent with non‐endocrine systemic toxicity. These were prevalent in the 9.0 and 27.2 μg Cu/L treatment groups but were minimally evident or absent in the 82.5 μg/L group, which was attributed to developmental arrest. In conclusion, developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. Developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. [ABSTRACT FROM AUTHOR]

Background: Glyphosate (GLY) is the most widely used herbicide in the world. Due to its mode of action as an inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, an important step in the shikimate pathway, specifically in plants, GLY is considered to be of low toxicity to non-target organisms. However, various studies have shown the negative effects of GLY on the mortality and development of different non-target organisms, including insects, rodents, fish and amphibians. To better understand the various effects of GLY in more detail, we studied the effects of GLY without co-formulants during the embryogenesis of the aquatic model organism Xenopus laevis. Results: A treatment with GLY affected various morphological endpoints in X. laevis tadpoles (body length, head width and area, eye area). Additionally, GLY interfered with the mobility as well as the neural and cardiac development of the embryos at stage 44/45. We were able to detect detailed structural changes in the cranial nerves and the heart and gained insights into the negative effects of GLY on cardiomyocyte differentiation. Conclusion: The application of GLY without co-formulants resulted in negative effects on several endpoints in the early embryonic development of X. laevis at concentrations that are environmentally relevant and concentrations that reflect the worst-case scenarios. This indicates that GLY could have a strong negative impact on the survival and lives of amphibians in natural waters. As a result, future GLY approvals should consider its impact on the environment.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host’s survival.

TRAAK is a membrane tension-activated K+ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the leak K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K+ channel.

The Western clawed frog Xenopus tropicalis is a diploid model system for both frog genetics and developmental biology, complementary to the paleotetraploid X. laevis. Here we report a chromosome-scale assembly of the X. tropicalis genome, improving the previously published draft genome assembly through the use of new assembly algorithms, additional sequence data, and the addition of a dense genetic map. The improved genome enables the mapping of specific traits (e.g., the sex locus or Mendelian mutants) and the characterization of chromosome-scale synteny with other tetrapods. We also report an improved annotation of the genome that integrates deep transcriptome sequence from diverse tissues and stages. The exon-intron structures of these genes are highly conserved relative to both X. laevis and human, as are chromosomal linkages ("synteny") and local gene order. A network analysis of developmental gene expression will aid future studies.

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.

Deleterious variants in SLC2A2 cause Fanconi-Bickel Syndrome (FBS), a glycogen storage disorder, whereas less common variants in SLC2A2 associate with numerous metabolic diseases. Phenotypic heterogeneity in FBS has been observed, but its causes remain unknown. Our goal was to functionally characterize rare SLC2A2 variants found in FBS and metabolic disease-associated variants to understand the impact of these variants on GLUT2 activity and expression and establish genotype-phenotype correlations. Complementary RNA-injected Xenopus laevis oocytes were used to study mutant transporter activity and membrane expression. GLUT2 homology models were constructed for mutation analysis using GLUT1, GLUT3, and XylE as templates. Seventeen FBS variants were characterized. Only c.457_462delCTTATA (p.Leu153_Ile154del) exhibited residual glucose uptake. Functional characterization revealed that only half of the variants were expressed on the plasma membrane. Most less common variants (except c.593 C>A (p.Thr198Lys) and c.1087 G>T (p.Ala363Ser)) exhibited similar GLUT2 transport activity as the wild type. Structural analysis of GLUT2 revealed that variants affect substrate-binding, steric hindrance, or overall transporter structure. The mutant transporter that is associated with a milder FBS phenotype, p.Leu153_Ile154del, retained transport activity. These results improve our overall understanding of the underlying causes of FBS and impact of GLUT2 function on various clinical phenotypes ranging from rare to common disease.

Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.