Revealing mitf functions and visualizing allografted tumor metastasis in colorless and immunodeficient Xenopus tropicalis.

Transparent immunodeficient animal models not only enhance in vivo imaging investigations of visceral organ development but also facilitate in vivo tracking of transplanted tumor cells. However, at present, transparent and immunodeficient animal models are confined to zebrafish, presenting substantial challenges for real-time, in vivo imaging studies addressing specific biological inquiries. Here, we employed a mitf-/-/prkdc-/-/il2rg-/- triple-knockout strategy to establish a colorless and immunodeficient amphibian model of Xenopus tropicalis. By disrupting the mitf gene, we observed the loss of melanophores, xanthophores, and granular glands in Xenopus tropicalis. Through the endogenous mitf promoter to drive BRAFV600E expression, we confirmed mitf expression in melanophores, xanthophores and granular glands. Moreover, the reconstruction of the disrupted site effectively reinstated melanophores, xanthophores, and granular glands, further highlighting the crucial role of mitf as a regulator in their development. By crossing mitf-/- frogs with prkdc-/-/il2rg-/- frogs, we generated a mitf-/-/prkdc-/-/il2rg-/- Xenopus tropicalis line, providing a colorless and immunodeficient amphibian model. Utilizing this model, we successfully observed intravital metastases of allotransplanted xanthophoromas and migrations of allotransplanted melanomas. Overall, colorless and immunodeficient Xenopus tropicalis holds great promise as a valuable platform for tumorous and developmental biology research.

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.

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies.

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

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.

Protocol for tail vein injection in Xenopus tropicalis tadpoles.

Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments. For complete details on the use and execution of this protocol, please refer to Kakebeen et al.,1 Patel et al.,2 and Patel et al.3.

Male-transmitted transgenerational effects of the herbicide linuron on DNA methylation profiles in Xenopus tropicalis brain and testis.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

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.

19th International Xenopus Conference Meeting Report: Latest developments and future perspectives.

The African clawed frog, Xenopus laevis, and the Western clawed frog, Xenopus tropicalis, have been foundational model organisms for establishing key principles of embryonic development. Today, the utility of Xenopus has been greatly expanded for studying a wide range of biological processes both in health and disease. Here, we describe the latest advancements from the Xenopus community, which span the molecular, cellular, tissue, and organismal scales.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic aging.

To address how conserved DNA methylation-based epigenetic aging is in diverse branches of the tree of life, we generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis) and built multiple epigenetic clocks. Dual species clocks were developed that apply to both humans and frogs (human-clawed frog clocks), supporting that epigenetic aging processes are evolutionary conserved outside mammals. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. We conclude that signatures of epigenetic aging are evolutionary conserved between frogs and mammals and that the associated genes relate to neural processes, altogether opening opportunities to employ Xenopus as a model organism to study aging.

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.

Molecular mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain.

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels.

Developmental expression of peroxiredoxin gene family in early embryonic development of Xenopus tropicalis.

Peroxidase genes (Prdx) encode a family of antioxidant proteins, which can protect cells from oxidative damage by reducing various cellular peroxides. This study investigated the spatiotemporal expression patterns of gene members in this family during the early development of Xenopus tropicalis. Real-time quantitative PCR showed that all members of this gene family have a distinct temporal expression pattern during the early development of X. tropicalis embryos. Additionally, whole mount in situ hybridization revealed that individual prdx genes display differential expression patterns, with overlapping expression in lymphatic vessels, pronephros, proximal tubule, and branchial arches. This study provides a basis for further study of the function of the prdx gene family.

Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

Steroid-receptor coactivator complexes in thyroid hormone-regulation of Xenopus metamorphosis.

Anuran metamorphosis is perhaps the most drastic developmental change regulated by thyroid hormone (T3) in vertebrate. It mimics the postembryonic development in mammals when many organs/tissues mature into adult forms and plasma T3 level peaks. T3 functions by regulating target gene transcription through T3 receptors (TRs), which can recruit corepressor or coactivator complexes to target genes in the absence or presence of T3, respectively. By using molecular and genetic approaches, we and others have investigated the role of corepressor or coactivator complexes in TR function during the development of two highly related anuran species, the pseudo-tetraploid Xenopus laevis and diploid Xenopus tropicalis. Here we will review some of these studies that demonstrate a critical role of coactivator complexes, particularly those containing steroid receptor coactivator (SRC) 3, in regulating metamorphic rate and ensuring the completion of metamorphosis.

Essential and subtype-dependent function of thyroid hormone receptors during Xenopus metamorphosis.

Thyroid hormone (T3) plays critical roles in organ metabolism and development in vertebrates. Anuran metamorphosis is perhaps the most dramatic and best studied developmental process controlled by T3. Many changes in different organs/tissues during anuran metamorphosis resemble the maturation/remodeling of the corresponding organs/tissues during mammalian postembryonic development. The plasma T3 level peaks during both anuran metamorphosis and mammalian postembryonic development. T3 exerts its developmental function through transcriptional regulation via T3 receptors (TRs). Studies on the metamorphosis of two highly related anurans, pseudo-tetraploid Xenopus laevis and diploid Xenopus tropicalis, have led to a dual function model for TRs during development. This has been supported by strong molecular and genetic evidence. Here we review some of the evidence with a focus on more recent gene knockout studies in Xenopus tropicalis. These studies have not only supported the model but also revealed novel and TR subtype-specific roles during Xenopus development, particularly a critical role of TRα in controlling developmental timing and rate.

The fast block to polyspermy breaks with convention.

JGP study (Komondor et al. 2023. J. Gen. Physiol. https://doi.org/10.1085/jgp.202213258) reveals that conventional PLC activation pathways are not required for the fertilization-induced depolarization of Xenopus eggs that prevents the entry of additional sperm.

Generation of translucent Xenopus tropicalis through triple knockout of pigmentation genes.

Amphibians generally have three types of pigment cells, namely, melanophores (black and brown), xanthophores (yellow and red), and iridophores (iridescent). Single knockout of the tyr, slc2a7, and hps6 genes in Xenopus tropicalis results in the absence of melanophores, xanthophores, and iridophores, respectively. The generation of triple- knockout (3KO) X. tropicalis for these three genes could allow for observation of internal organs without sacrificing the animals, which would be transparent due to the absence of pigments. In this study, we generated 3KO X. tropicalis, which is one of the most widely used model amphibians, through crossing of a slc2a7 single-knockout frog with a tyr and hps6 double-knockout frog, followed by intercrossing of their offspring. The 3KO tadpoles had transparent bodies like the nop mutant and the frogs had translucent bodies. This translucency allowed us to observe the heart, lungs, stomach, liver, and digestive tract through the ventral body skin without surgery. After intravital staining, 3KO X. tropicalis showed much clearer fluorescent signals of mineralized tissues compared with the wild type. These 3KO X. tropicalis provide a useful mutant line for continuous observation of internal organs and fluorescent signals in the body. In particular, such 3KO frogs would revolutionize fluorescence monitoring in transgenic tadpoles and frogs expressing fluorescent proteins.

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.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.