Midori Iida

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.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the peripheral nervous system of developing red seabream (Pagrus major).

We investigated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced effects on the morphology of peripheral nervous system (PNS) in the developing red seabream (Pagrus major) embryos. The embryos at 10h post-fertilization (hpf) were treated with 0, 0.1, 0.4 or 1.7 μg/L of TCDD in seawater for 80 min. The morphology of PNS was microscopically observed with florescence staining using an anti-acetylated tubulin antibody at 48, 78, 120 and 136 hpf. Axon length of facial nerve (VII) was found to be shortened by TCDD exposure. Axon guidance in the glossopharyngeal nerve (IX) and vagus nerve (X) was altered at 120 and 136 hpf in a TCDD dose-dependent manner. Lowest observable effect level of TCDD (0.1 μg/L) that induced the morphological alteration of PNS was lower than those of other endpoints on morphological deformities so far reported. Given that the growth cone at the tip of growing nerve axons advances under the influence of its surrounding tissues, we hypothesized that TCDD exposure would affect (1) the nerve cell proliferation/differentiation, (2) the structure of muscle as an axon target and (3) the nerve guidance factor in the embryos. By the immunostaining of embryos with an antibody against the neuronal specific RNA-binding protein, HuD, and an antibody against the sarcomeric myosin, no morphological effects were observed on the neural proliferation/differentiation and the structure of facial muscles of TCDD-treated embryos. In contrast, whole mount in situ hybridization of semaphorin 3A (Sema3A), a secretory axon repulsion factor, revealed the altered expression pattern of its transcripts in TCDD-treated embryos. Our findings suggest that TCDD treatment affects the projection of PNS in the developing red seabream embryos through the effects on the axonal growth cone guidance molecule such as Sema3A, but not on the neuronal differentiation/proliferation and axon target. The PNS in developing embryos may be one of the most sensitive biomarkers to the exposure of dioxin-like compounds.

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

ABSTRACT 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 TRβ, is indispensable for amphibian metamorphosis. However, specific roles for the TRβ isoform in metamorphosis are poorly understood. To address this issue, we generated trβ-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 trβ in metamorphosis. trβ-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trβ 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 trβ. Surprisingly, trβ-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRβ is not required but contributes to the timing of resorptive events of metamorphosis. Summary: Using knocked-out Xenopus tropicalis founders generated by CRISPR-Cas, TRβ was shown to have significant effects on developmental timing but is not required for completion of metamorphosis.

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.

Transient suppression of AHR activity in early red seabream embryos does not prevent the disruption of peripheral nerve projection by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The toxicity of dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mainly mediated by an aryl hydrocarbon receptor (AHR), which regulates the transcription of multiple target genes including cytochrome P450 1A (CYP1A). Our pervious study identified the presence of TCDD-induced defects of peripheral nerve projection in red seabream (Pagrus major) embryos. However, it remains unclear whether the TCDD-induced peripheral neurotoxicity is mediated by the AHR. To assess the contribution of the red seabream AHR (rsAHR) signaling pathway to the neuronal toxicity, red seabream embryos at 10h post-fertilization (hpf) were treated for 80 min with TCDD (0, 0.3, 5.3, and 37 nM in seawater) alone or in combination with CH223191 (500 nM in seawater), which is an AHR antagonist. A preliminary in vitro reporter gene assay confirmed that TCDD-induced transcriptional activity via rsAHR1 and rsAHR2 was suppressed by CH223191 treatment in a dose-dependent manner. CYP1A mRNA expression in embryos was determined by 2-step real time quantitative-polymerase chain reaction at 24 and 120 hpf and in situ hybridization at 48, 72, 96 and 120 hpf. The morphology of the peripheral nerve system (PNS) was also microscopically observed by fluorescent staining using an anti-acetylated tubulin antibody at 120 hpf. CYP1A mRNA expression was dose-dependently induced by TCDD at all of the examined developing stages. The suppression of TCDD-induced CYP1A expression by CH223191 treatment was observed in embryos at 24 and 48 hpf, while the effect of the rsAHR antagonist disappeared at 96 and 120 hpf. This phenomenon indicated the transient suppression of TCDD-induced rsAHR activation by CH223191 treatment. The immunostaining of peripheral nerves at 120 hpf demonstrated that the projections of the craniofacial nerve were altered in TCDD-treated embryos, and the frequency of TCDD-induced abnormal projections was not prevented by co-treatment with CH223191. These results indicate that the transient suppression of TCDD-induced rsAHR activation during the early developing stages of the red seabream does not influence the abnormal projection of peripheral nerves. In conclusion, transient rsAHR activation in the early stages of development is not involved in the neurotoxicity.

Nanosecond pulsed electric field incorporation technique to predict molecular mechanisms of teratogenicity and developmental toxicity of estradiol-17β on medaka embryos: DEVELOPMENTAL EFFECTS OF E2 ON MEDAKA EMBRYOS BY NSPEF INCORPORATION

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol‐17β (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.

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.

A network biology-based approach to evaluating the effect of environmental contaminants on human interactome and diseases

Environmental contaminant exposure can pose significant risks to human health. Therefore, evaluating the impact of this exposure is of great importance; however, it is often difficult because both the molecular mechanism of disease and the mode of action of the contaminants are complex. We used network biology techniques to quantitatively assess the impact of environmental contaminants on the human interactome and diseases with a particular focus on seven major contaminant categories: persistent organic pollutants (POPs), dioxins, polycyclic aromatic hydrocarbons (PAHs), pesticides, perfluorochemicals (PFCs), metals, and pharmaceutical and personal care products (PPCPs). We integrated publicly available data on toxicogenomics, the diseasome, protein-protein interactions (PPIs), and gene essentiality and found that a few contaminants were targeted to many genes, and a few genes were targeted by many contaminants. The contaminant targets were hub proteins in the human PPI network, whereas the target proteins in most categories did not contain abundant essential proteins. Generally, contaminant targets and disease-associated proteins were closely associated with the PPI network, and the closeness of the associations depended on the disease type and chemical category. Network biology techniques were used to identify environmental contaminants with broad effects on the human interactome and contaminant-sensitive biomarkers. Moreover, this method enabled us to quantify the relationship between environmental contaminants and human diseases, which was supported by epidemiological and experimental evidence. These methods and findings have facilitated the elucidation of the complex relationship between environmental exposure and adverse health outcomes.