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Molecular Phylogenetics and Evolution Jun 2024Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this...
Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this family are inconspicuously colored and considered non-aposematic. Epipedobates is among the youngest genus-level clades of Dendrobatidae that includes both aposematic and inconspicuous species. Using Sanger-sequenced mitochondrial and nuclear markers, we demonstrate deep genetic divergences among inconspicuous species of Epipedobates but relatively shallow genetic divergences among conspicuous species. Our phylogenetic analysis includes broad geographic sampling of the inconspicuous lineages typically identified as E. boulengeri and E. espinosai, which reveals two putative new species, one in west-central Colombia (E. sp. 1) and the other in north-central Ecuador (E. aff. espinosai). We conclude that E. darwinwallacei is a junior subjective synonym of E. espinosai. We also clarify the geographic distributions of inconspicuous Epipedobates species including the widespread E. boulengeri. We provide a qualitative assessment of the phenotypic diversity in each nominal species, with a focus on the color and pattern of inconspicuous species. We conclude that Epipedobates contains eight known valid species, six of which are inconspicuous. A relaxed molecular clock analysis suggests that the most recent common ancestor of Epipedobates is ∼11.1 million years old, which nearly doubles previous estimates. Last, genetic information points to a center of species diversity in the Chocó at the southwestern border of Colombia with Ecuador. A Spanish translation of this text is available in the supplementary materials.
Topics: Animals; Phylogeny; Poison Frogs; Anura; Mitochondria; Ecuador
PubMed: 38531492
DOI: 10.1016/j.ympev.2024.108065 -
Frontiers in Endocrinology 2024Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter...
Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T or T), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.
Topics: Animals; Humans; Xenopus laevis; Ecosystem; Thyroid Hormones; Thyroid Gland; Cell Proliferation
PubMed: 38529399
DOI: 10.3389/fendo.2024.1360188 -
Proceedings. Biological Sciences Mar 2024
PubMed: 38526482
DOI: 10.1098/rspb.2024.0528 -
Molecules and Cells Apr 2024A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic...
A comprehensive regulatory network of transcription factors controls the dorsoventral patterning of the body axis in developing vertebrate embryos. Bone morphogenetic protein signaling is essential for activating the Ventx family of homeodomain transcription factors, which regulates embryonic patterning and germ layer identity during Xenopus gastrulation. Although Ventx1.1 and Ventx2.1 of the Xenopus Ventx family have been extensively investigated, Ventx3.2 remains largely understudied. Therefore, this study aimed to investigate the transcriptional regulation of ventx3.2 during the embryonic development of Xenopus. We used goosecoid (Gsc) genome-wide chromatin immunoprecipitation-sequencing data to isolate and replicate the promoter region of ventx3.2. Serial deletion and site-directed mutagenesis were used to identify the cis-acting elements for Gsc and caudal type homeobox 1 (Cdx1) within the ventx3.2 promoter. Cdx1 and Gsc differentially regulated ventx3.2 transcription in this study. Additionally, positive cis-acting and negative response elements were observed for Cdx1 and Gsc, respectively, within the 5' flanking region of the ventx3.2 promoter. This result was corroborated by mapping the active Cdx1 response element (CRE) and Gsc response element (GRE). Moreover, a point mutation within the CRE and GRE completely abolished the activator and repressive activities of Cdx1 and Gsc, respectively. Furthermore, the chromatin immunoprecipitation-polymerase chain reaction confirmed the direct binding of Cdx1 and Gsc to the CRE and GRE, respectively. Inhibition of Cdx1 and Gsc activities at their respective functional regions, namely, the ventral marginal zone and dorsal marginal zone, reversed their effects on ventx3.2 transcription. These results indicate that Cdx1 and Gsc modulate ventx3.2 transcription in the ventral marginal zone and dorsal marginal zone by directly binding to the promoter region during Xenopus gastrulation.
Topics: Animals; Bone Morphogenetic Protein 4; Gastrula; Gene Expression Regulation, Developmental; Goosecoid Protein; Homeodomain Proteins; Promoter Regions, Genetic; Protein Binding; Transcription Factors; Transcription, Genetic; Xenopus laevis; Xenopus Proteins
PubMed: 38522664
DOI: 10.1016/j.mocell.2024.100058 -
The Journal of Biological Chemistry May 2024Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been...
Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar-protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.
Topics: Sodium-Glucose Transporter 1; Animals; Humans; Fluorometry; Xenopus laevis; Glucose; Oocytes; Protein Binding; Patch-Clamp Techniques; Galactose; Fructose; Binding Sites
PubMed: 38522518
DOI: 10.1016/j.jbc.2024.107215 -
The Journal of Biological Chemistry May 2024G-protein-gated inward rectifier K (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2,...
G-protein-gated inward rectifier K (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.
Topics: Animals; Rats; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Molecular Docking Simulation; Myocytes, Cardiac; Oocytes; Receptors, sigma; Sigma-1 Receptor; Xenopus laevis; Rats, Wistar
PubMed: 38522516
DOI: 10.1016/j.jbc.2024.107219 -
Biomedicine & Pharmacotherapy =... May 2024The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition...
The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition to their function as sensors that transduce diverse extracellular signals, including growth factors, fluid flow, and physical forces, cilia are intricately involved in cell cycle regulation and preservation of DNA integrity, as their formation and resorption dynamics are tightly linked to cell cycle progression. Recently, several studies have linked defects in specific ciliary proteins to the DNA damage response. However, it remains unclear whether and how primary cilia contribute to cancer development. Mebendazole (MBZ) is an anthelmintic drug with anticancer properties in some cancer cells. MBZ is continuously being tested for clinical studies, but the precise mechanism of its anticancer activities remains unknown. Here, using Xenopus laevis embryos as a model system, we discovered that MBZ significantly hinders cilia formation and induces DNA damage. Remarkably, primary cilium-bearing cancer cells exhibited heightened vulnerability to combined treatment with MBZ and conventional anticancer drugs. Our findings shed light on the specific influence of MBZ on cilia, rather than cytosolic microtubules, in triggering DNA damage, elucidating a previously unidentified mechanism underlying potential MBZ-mediated cancer therapy.
Topics: Cilia; DNA Damage; Animals; Mebendazole; Humans; Xenopus laevis; Antineoplastic Agents; Drug Synergism; Cell Line, Tumor; Embryo, Nonmammalian; Microtubules
PubMed: 38513592
DOI: 10.1016/j.biopha.2024.116434 -
Pflugers Archiv : European Journal of... May 2024Phosphate (Pi) is an essential nutrient, and its plasma levels are under tight hormonal control. Uphill transport of Pi into cells is mediated by the two Na-dependent Pi...
Phosphate (Pi) is an essential nutrient, and its plasma levels are under tight hormonal control. Uphill transport of Pi into cells is mediated by the two Na-dependent Pi transporter families SLC34 and SLC20. The molecular identity of a potential Pi export pathway is controversial, though XPR1 has recently been suggested by Giovannini and coworkers to mediate Pi export. We expressed XPR1 in Xenopus oocytes to determine its functional characteristics. Xenopus isoforms of proteins were used to avoid species incompatibility. Protein tagging confirmed the localization of XPR1 at the plasma membrane. Efflux experiments, however, failed to detect translocation of Pi attributable to XPR1. We tested various counter ions and export medium compositions (pH, plasma) as well as potential protein co-factors that could stimulate the activity of XPR1, though without success. Expression of truncated XPR1 constructs and individual domains of XPR1 (SPX, transmembrane core, C-terminus) demonstrated downregulation of the uptake of Pi mediated by the C-terminal domain of XPR1. Tethering the C-terminus to the transmembrane core changed the kinetics of the inhibition and the presence of the SPX domain blunted the inhibitory effect. Our observations suggest a regulatory role of XPR1 in cellular Pi handling rather than a function as Pi exporter. Accordingly, XPR1 senses intracellular Pi levels via its SPX domain and downregulates cellular Pi uptake via the C-terminal domain. The molecular identity of a potential Pi export protein remains therefore elusive.
Topics: Animals; Humans; Cell Membrane; Homeostasis; Oocytes; Phosphates; Xenopus laevis; Xenotropic and Polytropic Retrovirus Receptor
PubMed: 38507112
DOI: 10.1007/s00424-024-02941-0 -
Cell Apr 2024Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts...
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Topics: Cell Nucleolus; Cell Nucleus; Nuclear Proteins; Proton-Motive Force; RNA; Phase Separation; Intrinsically Disordered Proteins; Animals; Xenopus laevis; Oocytes
PubMed: 38503281
DOI: 10.1016/j.cell.2024.02.029 -
Disease Models & Mechanisms Jun 2024De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart...
De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.
Topics: Animals; Humans; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Heart; Heart Defects, Congenital; Phenotype; Protein Isoforms; Xenopus laevis; Xenopus Proteins
PubMed: 38501224
DOI: 10.1242/dmm.050507