With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments.

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and is associated with a number of potential outcomes, including impaired diastolic function, heart failure, and sudden cardiac death. Various etiologies have been described for HCM, including pressure overload and mutations in sarcomeric and non-sarcomeric genes. However, the molecular pathogenesis of HCM remains incompletely understood. In this study, we performed comparative transcriptome analysis to identify dysregulated genes common to five mouse HCM models of differing etiology: (i) mutation of myosin heavy chain 6, (ii) mutation of tropomyosin 1, (iii) expressing human phospholamban on a null background, (iv) knockout of frataxin, and (v) transverse aortic constriction. Gene-by-gene comparison identified five genes dysregulated in all five HCM models. Glutathione S-transferase kappa 1 (Gstk1) was significantly downregulated in the five models, whereas myosin heavy chain 7 (Myh7), connective tissue growth factor (Ctgf), periostin (Postn), and reticulon 4 (Rtn4) were significantly upregulated. Gene ontology comparison revealed that 51 cellular processes were significantly enriched in genes dysregulated in each transcriptome dataset. Among them, six processes (oxidative stress, aging, contraction, developmental process, cell differentiation, and cell proliferation) were related to four of the five genes dysregulated in all HCM models. GSTK1 was related to oxidative stress only, whereas the other four genes were related to all six cell processes except MYH7 for oxidative stress. Gene–gene functional interaction network analysis suggested correlative expression of GSTK1, MYH7, and actin alpha 2 (ACTA2). To investigate the implications of Gstk1 downregulation for cardiac function, we knocked out gstk1 in zebrafish using the clustered regularly interspaced short palindromic repeats/Cas9 system. We found that expression of the zebrafish homologs of MYH7, ACTA2, and actin alpha 1 were increased in the gstk1-knockout zebrafish. In vivo imaging of zebrafish expressing a fluorescent protein in cardiomyocytes showed that gstk1 deletion significantly decreased the end diastolic volume and, to a lesser extent, end systolic volume. These results suggest that downregulation of GSTK1 may be a common mechanism underlying HCM of various etiologies, possibly through increasing oxidative stress and the expression of sarcomere genes.

Both caloric restriction (CR) and resveratrol (RSV) have beneficial effects on obesity. However, the biochemical pathways that mediate these beneficial effects might be complex and interconnected and have not been fully elucidated. To reveal the common therapeutic mechanism of CR and RSV, we performed a comparative transcriptome analysis of adipose tissues from diet-induced obese (DIO) zebrafish and obese humans. We identified nine genes in DIO zebrafish and seven genes in obese humans whose expressions were regulated by CR and RSV. Although the gene lists did not overlap except for one gene, the gene ontologies enriched in the gene lists were highly overlapped, and included genes involved in adipocyte differentiation, lipid storage and lipid metabolism. Bioinformatic analysis of cis-regulatory sequences of these genes revealed that their transcriptional regulators also overlapped, including EP300, HDAC2, CEBPB, CEBPD, FOXA1, and FOXA2. We also identified 15 and 46 genes that were dysregulated in the adipose tissue of DIO zebrafish and obese humans, respectively. Bioinformatics analysis identified EP300, HDAC2, and CEBPB as common transcriptional regulators for these genes. EP300 is a histone and lysyl acetyltransferase that modulates the function of histone and various proteins including CEBPB, CEBPD, FOXA1, and FOXA2. We demonstrated that adiposity in larval zebrafish was significantly reduced by C646, an inhibitor of EP300 that antagonizes acetyl-CoA. The reduction of adiposity by C646 was not significantly different from that induced by RSV or co-treatment of C646 and RSV. These results indicate that the inhibition of EP300 might be a common therapeutic mechanism between CR and RSV in adipose tissues of obese individuals.

Sleep-wake states are impaired in various neurological disorders. Impairment of sleep-wake states can be an early condition that exacerbates these disorders. Therefore, treating sleep-wake dysfunction may prevent or slow the development of these diseases. Although many gene products are likely to be involved in the sleep-wake disturbance, hypnotics and psychostimulants clinically used are limited in terms of their mode of action and are not without side effects. Therefore, there is a growing demand for developing new hypnotics and psychostimulants with high efficacy and few side effects. Toward this end, animal models are indispensable for use in genetic and chemical screens to identify sleep-wake modifiers. As a proof-of-concept study, we performed behavioral profiling of zebrafish treated with chemical and genetic sleep-wake modifiers. We were able to demonstrate that behavioral profiling of zebrafish treated with hypnotics or psychostimulants from 9 to 10 days post-fertilization was sufficient to identify drugs with specific modes of action. We were also able to identify behavioral endpoints distinguishing GABA-A modulators and hypocretin (hcrt) receptor antagonists and between sympathomimetic and non-sympathomimetic psychostimulants. This behavioral profiling can serve to identify genes related to sleep-wake disturbance associated with various neuropsychiatric diseases and novel therapeutic compounds for insomnia and excessive daytime sleep with fewer adverse side effects.

Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish with another PPARα agonist, gemfibrozil, also increased expression of the mbp promoter-driven fluorescent reporter in an SREBF-dependent manner. These results suggest that activation of SREBFs by small molecular weight compounds may be a feasible therapeutic approach to stimulate myelination.