Network-based identification of diagnosis-specific trans-omic biomarkers via integration of multiple omics data.

The integration of multiple omics data promises to reveal new insights into the pathogenic mechanisms of complex human diseases, with the potential to identify avenues for the development of targeted therapies for disease subtypes. However, the extraction of diagnostic/disease-specific biomarkers from multiple omics data with biological pathway knowledge is a challenging issue in precision medicine. In this paper, we present a novel computational method to identify diagnosis-specific trans-omic biomarkers from multiple omics data. In the algorithm, we integrated multi-class sparse canonical correlation analysis (MSCCA) and molecular pathway analysis in order to derive discriminative molecular features that are correlated across different omics layers. We applied our proposed method to analyzing proteome and metabolome data of heart failure (HF), and extracted trans-omic biomarkers for HF subtypes; specifically, ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM). We were able to detect not only individual proteins that were previously reported from single-omics studies but also correlated protein-metabolite pairs characteristic of HF disease subtypes. For example, we identified hexokinase1(HK1)-d-fructose-6-phosphate as a paired trans-omic biomarker for DCM, which could significantly perturb amino-sugar metabolism. Our proposed method is expected to be useful for various applications in precision medicine.

Elucidating Key Characteristics of PFAS Binding to Human Peroxisome Proliferator-Activated Receptor Alpha: An Explainable Machine Learning Approach.

Per- and polyfluoroalkyl substances (PFAS) are widely employed anthropogenic fluorinated chemicals known to disrupt hepatic lipid metabolism by binding to human peroxisome proliferator-activated receptor alpha (PPARα). Therefore, screening for PFAS that bind to PPARα is of critical importance. Machine learning approaches are promising techniques for rapid screening of PFAS. However, traditional machine learning approaches lack interpretability, posing challenges in investigating the relationship between molecular descriptors and PPARα binding. In this study, we aimed to develop a novel, explainable machine learning approach to rapidly screen for PFAS that bind to PPARα. We calculated the PPARα-PFAS binding score and 206 molecular descriptors for PFAS. Through systematic and objective selection of important molecular descriptors, we developed a machine learning model with good predictive performance using only three descriptors. The molecular size (b_single) and electrostatic properties (BCUT_PEOE_3 and PEOE_VSA_PPOS) are important for PPARα-PFAS binding. Alternative PFAS are considered safer than their legacy predecessors. However, we found that alternative PFAS with many carbon atoms and ether groups exhibited a higher affinity for PPARα. Therefore, confirming the toxicity of these alternative PFAS compounds with such characteristics through biological experiments is important.

A key evolutionary step determining osmoregulatory ability for freshwater colonisation in early life stages of fish.

Colonisation of freshwater habitats by marine animals is a remarkable evolutionary event that has enriched biodiversity in freshwater ecosystems. The acquisition of tolerance to hypotonic stress during early life stages is presumed to be essential for their successful freshwater colonisation, but very little empirical evidence has been obtained to support this idea. This study aimed to comprehend the evolutionary changes in osmoregulatory mechanisms that enhance larval freshwater tolerance in amphidromous fishes, which typically spend their larval period in marine (ancestral) habitats and the rest of their life history stages in freshwater (derived) habitats. We compared the life history patterns and changes in larval survivorship and gene expression depending on salinity among three congeneric marine-originated amphidromous goby species (Gymnogobius), which had been suggested to differ in their larval dependence on freshwater habitats. An otolith microchemical analysis and laboratory-rearing experiment confirmed the presence of freshwater residents only in G. urotaenia and higher larval survivorship of this species in the freshwater condition than in the obligate amphidromous G. petschiliensis and G. opperiens. Larval whole-body transcriptome analysis revealed that G. urotaenia from both amphidromous and freshwater-resident populations exhibited the greatest differences in expression levels of several osmoregulatory genes, including aqp3, which is critical for water discharge from their body during early fish development. The present results consistently support the importance of enhanced freshwater tolerance and osmoregulatory plasticity in larval fish to establish freshwater forms, and further identified key candidate genes for larval freshwater adaptation and colonisation in the goby group.

CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.

Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.

Effects of exposure to oxytetracycline on the liver proteome of red seabream (Pagrus major) in a real administration scenario.

Oxytetracycline (OTC) is a widely used antibiotic in aquaculture. In this study, red seabream (Pagrus major), the most popular aquaculture species in Japan, were treated with OTC mimicking a real administration scenario in aquaculture. The treatment groups were as follows: no OTC, 40 mg/kg body wt/day (equivalent to the dose used in actual aquaculture), or 178 mg/kg body wt/day. The first exposure was conducted for a week (1st OTC exposure period), followed by a 4-week interval, and the second exposure was for one week (2nd OTC exposure period). We investigated the effects of OTC on the liver proteome with the isobaric tags for relative and absolute quantitation (iTRAQ) technology accompanied by liquid chromatography and mass spectrometry. The pathway and disease enrichment analyses of differentially abundant proteins in OTC-exposed groups compared to their respective controls showed that the abundance of proteins related to the immune and nervous systems was altered after the 1st and 2nd OTC exposures, respectively. Quantitative real-time PCR of the transcripts of immune-related genes corroborated with the results of proteome analysis. OTC exposure also modulated the expression of metabolism-related proteins after the 1st and 2nd OTC exposures. Furthermore, after four weeks of the 2nd exposure, weight loss and changes in the expression of proteins related to metabolism were observed, suggesting that OTC exposure disrupts the metabolic system and causes growth inhibition. Based on these results, we suggest that the use of OTC in aquaculture poses a health risk in fish species. Thus, we need to pay more attention to the contamination with OTC in aquaculture.

Prediction of single-cell mechanisms for disease progression in hypertrophic remodelling by a trans-omics approach.

Heart failure is a heterogeneous disease with multiple risk factors and various pathophysiological types, which makes it difficult to understand the molecular mechanisms involved. In this study, we proposed a trans-omics approach for predicting molecular pathological mechanisms of heart failure and identifying marker genes to distinguish heterogeneous phenotypes, by integrating multiple omics data including single-cell RNA-seq, ChIP-seq, and gene interactome data. We detected a significant increase in the expression level of natriuretic peptide A (Nppa), after stress loading with transverse aortic constriction (TAC), and showed that cardiomyocytes with high Nppa expression displayed specific gene expression patterns. Multiple NADH ubiquinone complex family, which are associated with the mitochondrial electron transport system, were negatively correlated with Nppa expression during the early stages of cardiac hypertrophy. Large-scale ChIP-seq data analysis showed that Nkx2-5 and Gtf2b were transcription factors characteristic of high-Nppa-expressing cardiomyocytes. Nppa expression levels may, therefore, represent a useful diagnostic marker for heart failure.

Network-based characterization of disease-disease relationships in terms of drugs and therapeutic targets.

MOTIVATION: Disease states are distinguished from each other in terms of differing clinical phenotypes, but characteristic molecular features are often common to various diseases. Similarities between diseases can be explained by characteristic gene expression patterns. However, most disease-disease relationships remain uncharacterized. RESULTS: In this study, we proposed a novel approach for network-based characterization of disease-disease relationships in terms of drugs and therapeutic targets. We performed large-scale analyses of omics data and molecular interaction networks for 79 diseases, including adrenoleukodystrophy, leukaemia, Alzheimer's disease, asthma, atopic dermatitis, breast cancer, cystic fibrosis and inflammatory bowel disease. We quantified disease-disease similarities based on proximities of abnormally expressed genes in various molecular networks, and showed that similarities between diseases could be explained by characteristic molecular network topologies. Furthermore, we developed a kernel matrix regression algorithm to predict the commonalities of drugs and therapeutic targets among diseases. Our comprehensive prediction strategy indicated many new associations among phenotypically diverse diseases. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.

Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide.

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.

Effects of prenatal bisphenol A exposure on the hepatic transcriptome and proteome in rat offspring.

Developmental exposure to bisphenol A (BPA) is associated with liver dysfunction and diseases in adulthood. The aims of this study were to assess the effects of prenatal BPA exposure on the hepatic transcriptome and proteome in female and male offspring and to understand adverse outcome pathways (AOPs) to observed phenotypic effects. Pregnant Wistar rats were exposed to 50 or 5000 µg BPA/kg bw/day, or 17ß-estradiol (E2, 50 µg/kg bw/day) from embryonic day 3 to 18. The liver transcriptome and proteome profiles were analyzed in the newborn (postnatal day 1; PND1) and weaning (PND21) rat offspring. Based on the differentially expressed genes/proteins derived from transcriptome and proteome profiles, we performed pathway, transcription factor, and disease enrichment analyses. A principal component analysis of transcriptome data demonstrated that prenatal BPA exposure caused masculinization of the hepatic transcriptome in females. Both of transcriptomic and proteomic data showed that prenatal BPA exposure led to the disruption of cell cycle, lipid homeostasis, and hormone balance in offspring. Most of the effects at the transcript level were extended from newborn to weaning in males, but were moderated until weaning in females. The alterations at the transcript and protein levels were accordant with the observation of increases in body weight and anogenital distance and changes in hepatosomatic index in the offspring. Collectively, we constructed AOPs with evidence of sex- and age-specific actions of prenatal BPA exposure in the offspring.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

Decomposing the effects of ocean environments on predator-prey body-size relationships in food webs.

Body-size relationships between predators and their prey are important in ecological studies because they reflect the structure and function of food webs. Inspired by studies on the impact of global warming on food webs, the effects of temperature on body-size relationships have been widely investigated; however, the impact of environmental factors on body-size relationships has not been fully evaluated because climate warming affects various ocean environments. Thus, here, we comprehensively investigated the effects of ocean environments and predator-prey body-size relationships by integrating a large-scale dataset of predator-prey body-size relationships in marine food webs with global oceanographic data. We showed that various oceanographic parameters influence prey size selection. In particular, oxygen concentration, primary production and salinity, in addition to temperature, significantly alter body-size relationships. Furthermore, we demonstrated that variability (seasonality) of ocean environments significantly affects body-size relationships. The effects of ocean environments on body-size relationships were generally remarkable for small body sizes, but were also significant for large body sizes and were relatively weak for intermediate body sizes, in the cases of temperature seasonality, oxygen concentration and salinity variability. These findings break down the complex effects of ocean environments on body-size relationships, advancing our understanding of how ocean environments influence the structure and functioning of food webs.

Effects of 4-Hydroxy-2,3,3',4',5-Pentachlorobiphenyl (4-OH-CB107) on Liver Transcriptome in Rats: Implication in the Disruption of Circadian Rhythm and Fatty Acid Metabolism.

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) have been detected in tissues of both wild animals and humans. Several previous studies have suggested adverse effects of OH-PCBs on the endocrine and nervous systems in mammals. However, there have been no studies on transcriptome analysis of the effects of OH-PCBs, and thus, the whole picture and mechanisms underlying the adverse effects induced by OH-PCBs are still poorly understood. We therefore investigated the mRNA expression profile in the liver of adult male Wistar rats treated with 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107) to explore the genes responsive to OH-PCBs and to understand the potential effects of the chemical. Next-generation RNA sequencing analysis revealed changes in the expression of genes involved in the circadian rhythm and fatty acid metabolism, such as nuclear receptor subfamily 1, group D, member 1, aryl hydrocarbon receptor nuclear translocator-like protein 1, cryptochrome circadian clock 1, and enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, in 4-OH-CB107-treated rats. In addition, biochemical analysis of the plasma revealed a dose-dependent increase in the leucine aminopeptidase, indicating the onset of liver damage. These results suggest that OH-PCB exposure may induce liver injury as well as disrupt the circadian rhythm and peroxisome proliferator-activated receptor-related fatty acid metabolism.

Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas.

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRß, is indispensable for amphibian metamorphosis. However, specific roles for the TRß isoform in metamorphosis are poorly understood. To address this issue, we generated trß-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of trß in metamorphosis. trß-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trß itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of trß Surprisingly, trß-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRß is not required but contributes to the timing of resorptive events of metamorphosis.

Fate of Worn-Out Functional Teeth in the Upper Jaw Dentition of Sicyopterus japonicus (Gobioidei: Sicydiinae) During Tooth Replacement.

Mochizuki and Fukui (Jpn J Ichthyol 30 () 27-36) studied the development and replacement of the upper jaw teeth in a Japanese fish species, Sicyopterus japonicus (Gobioidei: Sicydiinae), and they reported that worn-out functional teeth in the upper jaw were not shed outside the skin but were taken into the soft tissue of the upper jaw and completely resorbed there. To date, however, this phenomenon appears poorly documented. Furthermore, the mechanism for the resorption of these teeth remains to be determined. In this study, we examined this phenomenon by using 3D microcomputed tomography (m-CT), scanning electron microscopy (SEM), and various techniques of light (LM) and electron (EM) microcopy. This study demonstrated that the upper jaw dentition of this fish was more or less simultaneously replaced with the replacement occurring during short time periods and that the lingual movement of the replacement teeth to the functional tooth position advanced simultaneously in a given row. Furthermore, our study also revealed that many worn-out functional teeth were engulfed by the oral epithelium, invaginated into the lingual shallow ditch of the premaxilla, and were resorbed/degraded completely by numerous foreign body giant cells rather than by odontoclasts during periods of at least three intervals of tooth replacement. The complete resorption/degradation of worn-out functional teeth in the soft tissue of the upper jaw suggests the possibility of the reuse of their components (minerals such as Ca and P, including Fe) for rapid and successional production of new replacement teeth in the upper jaw of adult S. japonicus. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:111-124, 2018. © 2017 Wiley Periodicals, Inc.

Nanosecond pulsed electric field incorporation technique to predict molecular mechanisms of teratogenicity and developmental toxicity of estradiol-17ß on medaka embryos.

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol-17ß (E2 ) and predict the molecular mechanisms of teratogenicity and embryonic developmental defects caused by E2 on medaka (Oryzias latipes). The 5 hour post-fertilization embryos were exposed to co-treatment with 10 µm E2 and nsPEF for 2 hours and then continuously cultured under non-E2 and nsPEF conditions until hatching. Results documented that the time to hatching of embryos was significantly delayed in comparison to the control group and that typical abnormal embryo development, such as the delay of blood vessel formation, was observed. For DNA microarray analysis, 6 day post-fertilization embryos that had been continuously cultured under the non-E2 and nsPEF condition after 2 hour co-treatments were used. DNA microarray analysis identified 542 upregulated genes and one downregulated gene in the 6 day post-fertilization embryos. Furthermore, bioinformatic analyses using differentially expressed genes revealed that E2 exposure affected various gene ontology terms, such as response to hormone stimulus. The network analysis also documented that the estrogen receptor α in the mitogen-activated protein kinase signaling pathway may be involved in regulating several transcription factors, such as FOX, AKT1 and epidermal growth factor receptor. These results suggest that our nsPEF technique is a powerful tool for assessing teratogenicity and embryonic developmental toxicity of E2 and predict their molecular mechanisms in medaka embryos.

Strain differences in the proteome of dioxin-sensitive and dioxin-resistant mice treated with 2,3,7,8-tetrabromodibenzo-p-dioxin.

Dioxins cause various toxic effects through the aryl hydrocarbon receptor (AHR) in vertebrates, with dramatic species and strain differences in susceptibility. Although inbred mouse strains C3H/HeJ-lpr/lpr (C3H/lpr) and MRL/MpJ-lpr/lpr (MRL/lpr) are known as dioxin-sensitive and dioxin-resistant mice, respectively, the molecular mechanism underlying this difference remains unclear. The difference in the hepatic proteome of the two mouse strains treated with vehicle or 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) was investigated by a proteomic approach of two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF). To confirm the strain-difference in response to TBDD treatment, cytochrome P450 (CYP) 1A1 and 1A2 protein levels were measured in both strains. A dose of 10 µg/kg body weight of TBDD induced hepatic CYP1A1 and CYP1A2 expression in both strains, but the expression levels of both CYP1A proteins were higher in C3H/lpr mice than in MRL/lpr mice, supporting that C3H/lpr mice are more sensitive to dioxins than MRL/lpr mice. Proteins that were more induced or suppressed by TBDD treatment in C3H/lpr mice were successfully identified by 2-DE and MALDI-TOF/TOF, including proteins responsible for AHR activation through production of endogenous ligands such as aspartate aminotransferase, indolethylamine N-methyltransferase, and aldehyde dehydrogenases, as well as proteins reducing oxidative stress, such as superoxide dismutase and peroxiredoxins. Taken together, our results provide insights into the molecular mechanism underlying the high dioxin susceptibility of the C3H/lpr strain, in which AHR activation by TBDD is more prompted by the production of endogenous ligands, but the adaptation to oxidative stress is also acquired.

Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major).

The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5'-flanking regions of AHR1 and AHR2 genes. Both of the 5'-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5'-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5'-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5'-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.

Identification of aryl hydrocarbon receptor signaling pathways altered in TCDD-treated red seabream embryos by transcriptome analysis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces a broad spectrum of toxic effects including craniofacial malformation and neural damage in fish embryos. These effects are mainly mediated by the aryl hydrocarbon receptor (AHR). However, the mode of action between TCDD-induced AHR activation and adverse outcomes is not yet understood. To provide a comprehensive picture of the AHR signaling pathway in fish embryos exposed to TCDD, red seabream (Pagrus major) embryos were treated with graded concentrations of TCDD (0.3-37nM) in seawater, or with a mixture of TCDD and 500nM CH223191, an AHR-specific antagonist. The transcriptome of red seabream embryos was analyzed using a custom-made microarray with 6000 probes specifically prepared for this species. A Jonckheere-Terpstra test was performed to screen for genes that demonstrated altered mRNA expression levels following TCDD exposure. The signals of 1217 genes (as human homologs) were significantly altered in a TCDD concentration-dependent manner (q-value<0.2). Notably, the TCDD-induced alteration in mRNA expression was alleviated by co-exposure to CH223191, suggesting that the mRNA expression level of these genes was regulated by AHR. To identify TCDD-activated pathways, the microarray data were further subjected to gene set enrichment analysis (GSEA) and functional protein-protein interaction (PPI) network analysis. GSEA demonstrated that the effects of TCDD on sets of genes involved calcium, mitogen-activated protein kinase (MAPK), actin cytoskeleton, chemokine, T cell receptor, melanoma, vascular endothelial growth factor (VEGF), axon guidance, and renal cell carcinoma signaling pathways. These results suggest the hypotheses that TCDD induces immunosuppression via the calcium, MAPK, chemokine, and T cell receptor signaling pathways, neurotoxicity via VEGF signaling, and axon guidance alterations and teratogenicity via the dysregulation of the actin cytoskeleton and melanoma and renal cell carcinoma signaling pathways. Furthermore, the PPI network analysis indicated that the adverse outcome pathways of TCDD in the embryos might be propagated through several hub genes such as cell division control protein 42, phosphoinositide-3-kinase regulatory subunit 1, and guanine nucleotide-binding proteins. Understanding these pathways potentially allows for exploring the adverse outcome pathway of the effects of TCDD on the red seabream embryos.

Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.