An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

MicroRNAs in neural crest development and neurocristopathies.

The neural crest (NC) is a vertebrate-specific migratory population of multipotent stem cells that originate during late gastrulation in the region between the neural and non-neural ectoderm. This population of cells give rise to a range of derivatives, such as melanocytes, neurons, chondrocytes, chromaffin cells, and osteoblasts. Because of this, failure of NC development can cause a variety of pathologies, often syndromic, that are globally called neurocristopathies. Many genes are known to be involved in NC development, but not all of them have been identified. In recent years, attention has moved from protein-coding genes to non-coding genes, such as microRNAs (miRNA). There is increasing evidence that these non-coding RNAs are playing roles during embryogenesis by regulating the expression of protein-coding genes. In this review, we give an introduction to miRNAs in general and then focus on some miRNAs that may be involved in NC development and neurocristopathies. This new direction of research will give geneticists, clinicians, and molecular biologists more tools to help patients affected by neurocristopathies, as well as broadening our understanding of NC biology.

microRNAs associated with early neural crest development in Xenopus laevis.

BACKGROUND: The neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression. RESULTS: To uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells. CONCLUSION: We have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.

Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials.

Antibacterial resistance has become a serious crisis for world health over the last few decades, so that new therapeutic approaches are strongly needed to face the threat of resistant infections. Transcription factor decoys (TFD) are a promising new class of antimicrobial oligonucleotides with proven in vivo activity when combined with a bolaamphiphilic cationic molecule, 12-bis-THA. These two molecular species form stable nanoplexes which, however, present very scarce colloidal stability in physiological media, which poses the challenge of drug formulation and delivery. In this work, we reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which retains the ability to protect the oligonucleotide therapeutic from degradation and deliver it across the bacterial cell wall. We performed a physical-chemical study to investigate how the incorporation of 12-bis-THA and TFD affects the structure of POPC- and POPC/DOPE liposomes. Analysis was performed using dynamic light scattering (DLS), ζ-potential measurements, small-angle x-ray scattering (SAXS), and steady-state fluorescence spectroscopy to better understand the structure of the liposomal formulations containing the 12-bis-THA/TFD complexes. Oligonucleotide delivery to model Escherichia coli bacteria was assessed by means of confocal scanning laser microscopy (CLSM), evidencing the requirement of a fusogenic helper lipid for transfection. Preliminary biological assessments suggested the necessity of further development by modulation of 12-bis-THA concentration in order to optimize its therapeutic index, i.e. the ratio of antibacterial activity to the observed cytotoxicity. In summary, POPC/DOPE/12-bis-THA liposomes appear as promising formulations for TFD delivery.

myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

DHODH modulates transcriptional elongation in the neural crest and melanoma.

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.

Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells.

In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3ß) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3ß, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3ß-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3ß by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.

Klhl31 attenuates ß-catenin dependent Wnt signaling and regulates embryo myogenesis.

Klhl31 is a member of the Kelch-like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, carboxy-terminal Kelch repeats and a central linker region (Back domain). In developing somites Klhl31 is highly expressed in the myotome downstream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis-expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20). The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with ß-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or ß-catenin. Klhl31 mis-expression in the developing neural tube affected its dorso-ventral patterning and led to reduced dermomyotome and myotome size. Co-transfection of a Wnt3a expression vector with Klhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.

Determining miRNA Expression Patterns in Xenopus.

Whole-mount in situ hybridization (WISH) is a technique that enables temporal and spatial visualization of RNA molecules in an embryo or whole tissue by using a complementary labelled probe. MicroRNAs are short noncoding RNAs of 20-25 nt in length mainly involved in posttranscriptional regulation of gene expression. In this chapter, we describe how to visualize miRNAs in Xenopus laevis or tropicalis by WISH using two different approaches: LNA-WISH to visualize mature miRNAs and pri-miRNA-WISH to visualize the immature form of miRNAs, the pri-miRNAs.

An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly utilized to study gene function. MicroRNAs (miRNAs) are short noncoding RNAs, 20-25 nucleotides long, which affect gene expression through posttranscriptional repression. As miRNAs are so small and due to the limitations of known PAM sequences, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore developed a novel approach using two guide RNAs to effectively "drop out" a miRNA. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of q-RT-PCR and Sanger sequencing. To show specificity of the phenotype, we provide a protocol to use miRNA mimics to rescue the KO phenotype.

Wnt/ß-catenin signalling is required for pole-specific chromatin remodeling during planarian regeneration.

For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/ß-catenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation, the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a Wnt/ß-catenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct Wnt/ß-catenin targets, which trigger the expression of posterior effectors. Finally, we identify FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer region.

Xenopus: an ideal system for chemical genetics.

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.

Expression analysis of chick Frizzled receptors during spinal cord development.

Frizzleds (Fzds) are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless gene. FZDs are critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. Here, we preformed a comprehensive expression profile of Wnt receptors (FZD) by using situ hybridization to identify FZDs that are expressed in dorsal-ventral regions of the neural tube development. Our data show specific expression for FZD1,2,3,7,9 and 10 in the chick developing spinal cord. This expression profile of cFZD receptors offers the basis for functional studies in the future to determine roles for the different FZD receptors and their interactions with Wnts during dorsal-ventral neural tube development in vivo. Furthermore, we also show that co-overexpression of Wnt1/3a by in vivo electroporation affects FZD7/10 expression in the neural tube. This illustrates an example of Wnts-FZDs interactions during spinal cord neurogenesis.

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.

Frizzled-10 promotes sensory neuron development in Xenopus embryos.

Formation of the vertebrate nervous system requires coordinated cell-cell interactions, intracellular signalling events, gene transcription, and morphogenetic cell movements. Wnt signalling has been involved in regulating a wide variety of biological processes such as embryonic patterning, cell proliferation, cell polarity, motility, and the specification of cell fate. Wnt ligands associate with their receptors, members of the frizzled family (Fz). In Xenopus, five members of the frizzled family are expressed in the early nervous system. We have investigated the role of Xenopus frizzled-10 (Fz10) in neural development. We show that Fz10 is expressed in the dorsal neural ectoderm and neural folds in the region where primary sensory neurons develop. Fz10 mediates canonical Wnt signalling and interacts with Wnt1 and Wnt8 but not Wnt3a as shown in synergy assays. We find that Fz10 is required for the late stages of sensory neuron differentiation. Overexpression of Fz10 in Xenopus leads to an increase in the number of sensory neurons. Loss of Fz10 function using morpholinos inhibits the development of sensory neurons in Xenopus at later stages of neurogenesis and this can be rescued by co-injection of modified Fz10B and beta-catenin. In mouse P19 cells induced by retinoic acid to undergo neural differentiation, overexpression of Xenopus Fz10 leads to an increase in the number of neurons generated while siRNA knockdown of endogenous mouse Fz10 inhibits neurogenesis. Thus we propose Fz10 mediates Wnt1 signalling to determine sensory neural differentiation in Xenopus in vivo and in mouse cell culture.

In Vivo Assessment of Drug-Induced Hepatotoxicity Using Xenopus Embryos.

Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in X. laevis embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. Xenopus embryos have been used as a toxicity model in the frog embryo teratogenesis assay on Xenopus (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using Xenopus embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, Xenopus embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on Xenopus embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in Xenopus embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development.

FZD10 regulates cell proliferation and mediates Wnt1 induced neurogenesis in the developing spinal cord.

Wnt/FZD signalling activity is required for spinal cord development, including the dorsal-ventral patterning of the neural tube, where it affects proliferation and specification of neurons. Wnt ligands initiate canonical, ß -catenin-dependent, signaling by binding to Frizzled receptors. However, in many developmental contexts the cognate FZD receptor for a particular Wnt ligand remains to be identified. Here, we characterized FZD10 expression in the dorsal neural tube where it overlaps with both Wnt1 and Wnt3a, as well as markers of dorsal progenitors and interneurons. We show FZD10 expression is sensitive to Wnt1, but not Wnt3a expression, and FZD10 plays a role in neural tube patterning. Knockdown approaches show that Wnt1 induced ventral expansion of dorsal neural markes, Pax6 and Pax7, requires FZD10. In contrast, Wnt3a induced dorsalization of the neural tube is not affected by FZD10 knockdown. Gain of function experiments show that FZD10 is not sufficient on its own to mediate Wnt1 activity in vivo. Indeed excess FZD10 inhibits the dorsalizing activity of Wnt1. However, addition of the Lrp6 co-receptor dramatically enhances the Wnt1/FZD10 mediated activation of dorsal markers. This suggests that the mechanism by which Wnt1 regulates proliferation and patterning in the neural tube requires both FZD10 and Lrp6.

The MH1 domain of Smad3 interacts with Pax6 and represses autoregulation of the Pax6 P1 promoter.

Pax6 transcription is under the control of two main promoters (P0 and P1), and these are autoregulated by Pax6. Additionally, Pax6 expression is under the control of the TGFbeta superfamily, although the precise mechanisms of such regulation are not understood. The effect of TGFbeta on Pax6 expression was studied in the FHL124 lens epithelial cell line and was found to cause up to a 50% reduction in Pax6 mRNA levels within 24 h. Analysis of luciferase reporters showed that Pax6 autoregulation of the P1 promoter, and its induction of a synthetic promoter encoding six paired domain-binding sites, were significantly repressed by both an activated TGFbeta receptor and TGFbeta ligand stimulation. Subsequently, a novel Pax6 binding site in P1 was shown to be necessary for autoregulation, indicating a direct influence of Pax6 protein on P1. In transfected cells, and endogenously in FHL124 cells, Pax6 co-immunoprecipitated with Smad3 following TGFbeta receptor activation, while in GST pull-down experiments, the MH1 domain of Smad3 was observed binding the RED sub-domain of the Pax6 paired domain. Finally, in DNA adsorption assays, activated Smad3 inhibited Pax6 from binding the consensus paired domain recognition sequence. We hypothesize that the Pax6 autoregulatory loop is targeted for repression by the TGFbeta/Smad pathway, and conclude that this involves diminished paired domain DNA-binding function resulting from a ligand-dependant interaction between Pax6 and Smad3.

Paracetamol-induced liver injury modelled in Xenopus laevis embryos.

INTRODUCTION: Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC). METHOD: Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (1) microRNA-122 (miR-122) expression (a hepatic marker), (2) free GSH concentration (a marker of oxidative stress) and (3) NAC antioxidant intervention. RESULTS: The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had a significantly higher amount of GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively). CONCLUSION: Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research.