Use Of Xenopus Research Articles

SAT013 Characterization Of CRISPR/Cas9 Induced RXR Isotype Mutants In Xenopus Laevis

Abstract Disclosure: J.D. Furlow: None. S. Saini: None. B.J. Mengeling: None. M.E. Horb: None. Retinoid-X receptors (RXRs) occupy a unique position in the nuclear receptor (NR) superfamily. While some actions of the highly conserved RXRs may be independent, RXRs most often are heterodimer partners for several NRs that regulate development, reproduction and metabolism. As such, environmental chemicals interfering with the activity of RXRs and their partners may have significant deleterious effects on human health, often in developmental window, cell-type, and gene selective ways. The frog Xenopus has long served as an excellent model for studying vertebrate development including thyroid hormone action, and recent advances in genome editing have greatly enhanced its utility for investigating basic cell and organismal biology as well as human disease processes. To determine isotype specific roles in response to natural and synthetic ligands as well as candidate environmental chemicals interacting with RXRs in vivo, we created targeted germline mutations in the Xenopus tropicalis RXRα (rxra), RXRβ (rxrb), and RXRγ (rxrg) genes. For CRISPR/Cas9 mediated genome editing, guide RNAs were designed to target sequences just prior to or within the first RXR DNA binding domains. We raised all three RXR homozygous null mutants up to and through metamorphosis. The rxrb and rxrg homozygous null mutants completed metamorphosis and allowed us to establish adult null lines, but the rxra homozygous null mutants die at metamorphic climax, reminiscent of the thyroid hormone receptor (thra/thrb) double mutants. Some rxra mutant froglets also lack forearms or have forelimb defects, implying a role for rxra in RA mediated forelimb specification. The X. tropicalis rxrg homozygous null mutants show a trend toward an increased rate of metamorphosis versus matched wild-type controls, with significantly elevated tshb gene expression as newly metamorphosed froglets suggesting impaired negative feedback by TH. Significantly altered opsin gene expression in the froglet eye was also observed in rxrg mutant retinas: elevated short wave opsins opn1sw1 and opn1sw2 and sharply reduced long wave opsin opn1lw. This elevated tshb and opn1sw1 expression is highly similar to what is found in mouse rxrg null mutants; thus RXRγ action in the eye and pituitary appears to be highly conserved between mammals and amphibians. The establishment of these new RXR isotype mutant specific lines will support the expanded use of Xenopus to study basic biology and environmental toxicology involving TR-RXR and other NR-RXR partnerships. Presentation: Saturday, June 17, 2023

Imaging Structural and Functional Dynamics in Xenopus Neurons.

In vivo time-lapse imaging has been a fruitful approach to identify structural and functional changes in the Xenopus nervous system in tadpoles and adult frogs. Structural imaging studies have identified fundamental aspects of brain connectivity, development, plasticity, and disease and have been instrumental in elucidating mechanisms regulating these events in vivo. Similarly, assessment of nervous system function using dynamic changes in calcium signals as a proxy for neuronal activity has demonstrated principles of neuron and circuit function and principles of information organization and transfer within the brain of living animals. Because of its many advantages as an experimental system, use of Xenopus has often been at the forefront of developing these imaging methods for in vivo applications. Protocols for in vivo structural and functional imaging-including cellular labeling strategies, image collection, and image analysis-will expand the use of Xenopus to understand brain development, function, and plasticity.

Xenopus as a platform for discovery of genes relevant to human disease.

Congenital birth defects result from an abnormal development of an embryo and have detrimental effects on children's health. Specifically, congenital heart malformations are a leading cause of death among pediatric patients and often require surgical interventions within the first year of life. Increased efforts to navigate the human genome provide an opportunity to discover multiple candidate genes in patients suffering from birth defects. These efforts, however, fail to provide an explanation regarding the mechanisms of disease pathogenesis and emphasize the need for an efficient platform to screen candidate genes. Xenopus is a rapid, cost effective, high-throughput vertebrate organism to model the mechanisms behind human disease. This review provides numerous examples describing the successful use of Xenopus to investigate the contribution of patient mutations to complex phenotypes including congenital heart disease and heterotaxy. Moreover, we describe a variety of unique methods that allow us to rapidly recapitulate patients' phenotypes in frogs: gene knockout and knockdown strategies, the use of fate maps for targeted manipulations, and novel imaging modalities. The combination of patient genomics data and the functional studies in Xenopus will provide necessary answers to the patients suffering from birth defects. Furthermore, it will allow for the development of better diagnostic methods to ensure early detection and intervention. Finally, with better understanding of disease pathogenesis, new treatment methods can be tailored specifically to address patient's phenotype and genotype.

Xenopus in revealing developmental toxicity and modeling human diseases.

The Xenopus model offers many advantages for investigation of the molecular, cellular, and behavioral mechanisms underlying embryo development. Moreover, Xenopus oocytes and embryos have been extensively used to study developmental toxicity and human diseases in response to various environmental chemicals. This review first summarizes recent advances in using Xenopus as a vertebrate model to study distinct types of tissue/organ development following exposure to environmental toxicants, chemical reagents, and pharmaceutical drugs. Then, the successful use of Xenopus as a model for diseases, including fetal alcohol spectrum disorders, autism, epilepsy, and cardiovascular disease, is reviewed. The potential application of Xenopus in genetic and chemical screening to protect against embryo deficits induced by chemical toxicants and related diseases is also discussed.

Exome Sequencing Implicates SWI/SNF Chromatin Remodeling Genes in Human Congenital Hydrocephalus

Abstract INTRODUCTION Congenital Hydrocephalus (CH) affects 1 in 1000 live births and is treated with lifelong surgical cerebrospinal fluid (CSF) diversion with substantial morbidity. Our group recently sequenced 180 probands with CH and identified 4 novel CH genes, all of which regulate neural stem cell (NSC) fate. Nonetheless, these genes account for just 10% of studied cases. METHODS We doubled the size of our cohort through robust recruitment and exome-sequenced 361 CH probands, including 216 case-parent trios. To study the effect of novel mutations on brain development, we developed a Xenopus tropicalis model of CH using CRISPR/Cas9. Multiple, nonoverlapping CRISPR guide RNAs targeted against candidate genes were designed and injected into fertilized single cell Xenopus embryos via microinjection. Ventricular morphology and CSF flow dynamics were analyzed via optical coherence tomography (OCT) imaging. Histological sections from WT and mutant Xenopus embryos were subjected to whole-mount in Situ hybridization to assess expression of various markers of NSC growth and proliferation. RESULTS We identified new causative mutations in both previously identified and novel genes, all of which regulate ventricular zone NSC fate. Of these, enrichment in rare, damaging variants was highest in the SWI/SNF chromatin remodeling complex genes SMARCC1 and ARID1B (P = 1.83 × 10-9). Xenopus tadpoles harboring mutations in SMARCC1 exhibited marked ventriculomegaly, aqueductal stenosis, and impaired CSF flow. In Situ hybridization of SMARCC1 knockout brain sections demonstrated decreased expression of genes regulating NSC growth and proliferation. Notably, injection of wild type SMARCC1 RNA restored normal brain development in SMARCC1 KO embryos. CONCLUSION These findings implicate SWI/SNF complex genes in CH and validate the use of Xenopus for investigating additional candidate genes. These findings highlight the importance of NSC dysregulation in CH pathology, and suggest that in a subset of patients the risk of adverse neurodevelopmental outcomes may be unaltered whether or not shunting is performed.

Abstract SY28-02: A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma

Abstract Neuroblastoma is associated with stalling of normal differentiation, resulting in excessive proliferation of neuroblastic precursors. Here we show that Xenopus frog embryos can be used as a rapid and versatile model to investigate the molecular regulation of proliferation versus differentiation of neuroblasts and what may go wrong with this in neuroblastoma. We find that CDK-dependent phosphorylation of the transcriptional master regulator ASCL1 plays a critical role in controlling the balance between cell proliferation and differentiation in embryonic neuroblasts and in neuroblastoma, and can be modulated to reactivate differentiation of neuroblastoma cells. ASCL1 is a master regulator proneural transcription factor that controls the balance between proliferation and differentiation of neuroblasts and is expressed transiently during formation of noradrenergic neurons arising from the neural crest. Embryos of the frog Xenopus laevis provide a versatile, rapid, and inexpensive system to study the role of ASCL1 phosphorylation by cyclin-dependent kinases in restraining neuronal differentiation. We found that enhanced cyclin-dependent kinase activity or elevated MYCN levels are sufficient to lock neuroblasts into a noradrenergic precursor-like state by multisite phosphorylation of ASCL1 protein. Conversely, preventing phosphorylation of ASCL1 can potentiate neuronal differentiation even in the face of proproliferative cues. Thus, the in vivo use of Xenopus as a model organism led us to the hypothesis that neuroblastoma arises from neuroblasts that fail to undergo the normal developmental program of differentiation due to precocious phosphorylation of ASCL1. We have gone on to test this hypothesis directly in neuroblastoma cells. We show that ASCL1 is phosphorylated by cyclin-dependent kinases and that phospho-ASCL1 supports the proproliferative program in neuroblastoma cells. However, preventing CDK-dependent phosphorylation of ASCL1 results in changes in the genome-wide transcriptional program of neuroblastoma cells, leading to suppression of proproliferative targets and simultaneous activation of genes that drive cell cycle exit and differentiation. Mechanistically, ASCL1 ChIPSeq reveals enhanced binding of un(der)phosphorylated ASCL1 at sites associated with prodifferentiation targets. Moreover, PHOX protein binding at key downstream regulatory elements is also modulated by ASCL1 phosphorylation. Finally, we also show that chemical CDK inhibition is sufficient to drive differentiation of neuroblastoma cells in a manner dependent on endogenous ASCL1. Therefore, we conclude that CDK-dependent phosphorylation of ASCL1 acts as a critical fulcrum controlling the balance between proliferation and differentiation in Xenopus and in human cells, and thus offers a novel therapeutic opportunity for neuroblastoma. Citation Format: Anna Philpott, Fahad Ali, Luke Wylie, Daniel Marcos Corchado, Igor Chernukhin, Evangelia Papachristou, Tatiana Papkovskaia, Liam Lee, Christopher Weekes, Tom Hiscock, Clive D'Santos, Suzanne Turner, Jason Carroll. A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY28-02.

Evolution of ligands, receptors and metabolizing enzymes of thyroid signaling

Thyroid hormones (THs) play important roles in vertebrates such as the control of the metabolism, development and seasonality. Given the pleiotropic effects of thyroid disorders (developmental delay, mood disorder, tachycardia, etc), THs signaling is highly investigated, specially using mammalian models. In addition, the critical role of TH in controlling frog metamorphosis has led to the use of Xenopus as another prominent model to study THs action. Nevertheless, animals regarded as non-model species can also improve our understanding of THs signaling. For instance, studies in amphioxus highlighted the role of Triac as a bona fide thyroid hormone receptor (TR) ligand. In this review, we discuss our current understanding of the THs signaling in the different taxa forming the metazoans (multicellular animals) group. We mainly focus on three actors of the THs signaling: the ligand, the receptor and the deiodinases, enzymes playing a critical role in THs metabolism. By doing so, we also pinpoint many key questions that remain unanswered. How can THs accelerate metamorphosis in tunicates and echinoderms while their TRs have not been yet demonstrated as functional THs receptors in these species? Do THs have a biological effect in insects and cnidarians even though they do not have any TR? What is the basic function of THs in invertebrate protostomia? These questions can appear disconnected from pharmacological issues and human applications, but the investigation of THs signaling at the metazoans scale can greatly improve our understanding of this major endocrinological pathway.

Using Xenopus to understand human disease and developmental disorders.

Model animals are crucial to biomedical research. Among the commonly used model animals, the amphibian, Xenopus, has had tremendous impact because of its unique experimental advantages, cost effectiveness, and close evolutionary relationship with mammals as a tetrapod. Over the past 50years, the use of Xenopus has made possible many fundamental contributions to biomedicine, and it is a cornerstone of research in cell biology, developmental biology, evolutionary biology, immunology, molecular biology, neurobiology, and physiology. The prospects for Xenopus as an experimental system are excellent: Xenopus is uniquely well-suited for many contemporary approaches used to study fundamental biological and disease mechanisms. Moreover, recent advances in high throughput DNA sequencing, genome editing, proteomics, and pharmacological screening are easily applicable in Xenopus, enabling rapid functional genomics and human disease modeling at a systems level.

Xenopus embryonic epidermis as a mucociliary cellular ecosystem to assess the effect of sex hormones in a non-reproductive context

BackgroundHow important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct.ResultsWe showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of “non-typical” cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin.ConclusionsThis study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.

Strategies for characterising cis-regulatory elements in Xenopus

Understanding the cis-regulatory architecture of metazoan organisms is the greatest challenge facing genome biology today. In vertebrate organisms, distinct sequence elements mediate transcriptional regulation and are scattered throughout the genome, either proximal or distal to promoters. The identification of transcriptional enhancers has proven rather difficult by conventional experimental approaches. In the past decade, the rapid generation of genomic sequences for multiple vertebrate organisms, accompanied by sophisticated comparative tools, has facilitated the identification of non-coding evolutionarily conserved regions that may encode cis-regulatory elements. Validating computational predictions and characterising cis-regulatory elements in vivo, however, has been a major bottleneck, mainly because the most commonly used organism for these experiments has been the mouse, and generating transgenic mice or modifying the mouse genome continues to be a labour-intensive, low-throughput, expensive process. This has led to the use of Xenopus, which holds great promise for high-throughput interrogation of putative cis-regulatory elements. In particular, Xenopus tropicalis may become particularly powerful for elucidating regulatory networks, chiefly because it is amenable to genetic manipulations, and its genome is being sequenced.

John Gurdon

John Gurdon

Nuclear Assembly, Structure, and Function: The Use of Xenopus in Vitro Systems

Nuclear Assembly, Structure, and Function: The Use of Xenopus in Vitro Systems

XENOPUS LAEVIS AND DEVELOPMENTAL BIOLOGY

XENOPUS LAEVIS AND DEVELOPMENTAL BIOLOGY

PHYSIOLOGY OF EGG EXTRUSION IN FEMALE XENOPUS FROG TEST FOR PREGNANCY11

IN 1930, HOGBEN (1) first showed that anterior pituitary lobe extracts could cause external deposition of ova in the hypophysectomised mature female Xenopus laevis (the South African clawed frog). Following this original observation Hog-ben, Charles and Slome (2), Shapiro and Zwarenstein (3, 4), and Bellerby (5, 6) cor-roborated Hogben's finding. Hence the use of the female Xenopus as an assay animal for gonadotropic substances came into use in South Africa. In 1934, Shapiro and Zwarenstein (7, 8) and Bellerby (9) working independently, simultaneously suggested the use of the Xenopus frog reaction as a test for gonado-tropic hormones in pregnancy urines. Many confirmatory papers on this simple test for pregnancy were soon forthcoming (10–22). This characteristic extrusion of eggs by the female Xenopus is rapidly becoming widely used as a quick test for the early diagnosis of pregnancy.