Globin Digest Improves Visceral Adiposity Through UCP1 Upregulation in Diet-Induced Obese Zebrafish and Mice.

Globin digest (GD), a bioactive oligopeptide derived from porcine hemoglobin proteins, has been demonstrated to have beneficial effects on improving postprandial hyperlipidemia, hyperglycemia, and liver injury. We previously reported the lipid-lowering effects of GD using a zebrafish obesogenic test. Here, we sought to evaluate the effect of GD on visceral adiposity and the underlying molecular mechanisms using zebrafish and mouse obesity models. GD ameliorated dyslipidemia and suppressed the accumulation of visceral adipose tissue (VAT) in adult obese zebrafish. Transcriptomic analysis by RNA sequencing of GD-treated adult zebrafish revealed that GD upregulated UCP1-related pathways. Further, we performed mouse experiments and found that GD intake (2 mg/g body weight/day) was associated with lowered plasma triglyceride and total cholesterol levels, decreased VAT accumulation, and improved adipocyte hypertrophy with the upregulation of Ucp1 expression in white adipose tissue at both the mRNA and protein levels. Taken together, these results indicate that GD improves visceral adiposity by upregulating UCP1 expression, providing a novel perspective on combating obesity.

Preventive Effects of Green Tea Extract against Obesity Development in Zebrafish.

Various natural products (NPs) have been used to treat obesity and related diseases. However, the best way to fight obesity is preventive, with accurate body weight management through exercise, diet, or bioactive NPs to avoid obesity development. We demonstrated that green tea extract (GTE) is an anti-obesity NP using a zebrafish obesity model. Based on a hypothesis that GTE can prevent obesity, the objective of this study was to assess GTE's ability to attenuate obesity development. Juvenile zebrafish were pretreated with GTE for seven days before obesity induction via a high-fat diet; adult zebrafish were pretreated with GTE for two weeks before obesity induction by overfeeding. As a preventive intervention, GTE significantly decreased visceral adipose tissue accumulation in juveniles and ameliorated visceral adiposity and plasma triglyceride levels in adult zebrafish obesity models. RNA sequencing analysis was performed using liver tissues from adult obese zebrafish, with or without GTE administration, to investigate the underlying molecular mechanism. Transcriptome analysis revealed that preventive GTE treatment affects several pathways associated with anti-obesity regulation, including activation of STAT and downregulation of CEBP signaling pathways. In conclusion, GTE could be used as a preventive agent against obesity.

RNA-seq Based Transcriptome Analysis of the Anti-Obesity Effect of Green Tea Extract Using Zebrafish Obesity Models.

Green tea is a popular beverage that is rich in polyphenolic compounds such as catechins. Its major content, (-)-epigallocatechin-3-gallate, has been shown to have beneficial effects on several diseases including cancer, metabolic syndrome, cardiovascular diseases, and neurodegenerative diseases. The aim of this study was to assess the anti-obesity effects and the underlying molecular mechanisms of green tea extract (GTE) using zebrafish larva and adult obesity models. We administered 100 µg/mL GTE to zebrafish larvae and performed a short-term obesogenic test. GTE significantly decreased the visceral adipose tissue volume induced by a high-fat diet. Oral administration (250 µg/g body weight/day) of GTE to adult diet-induced obese zebrafish also significantly reduced their visceral adipose tissue volume, with a reduction of plasma triglyceride and total cholesterol levels. To investigate the molecular mechanism underlying the GTE effects, we conducted RNA sequencing using liver tissues of adult zebrafish and found that GTE may ameliorate the obese phenotypes via the activation of Wnt/ß-catenin and adenosine monophosphate-activated protein kinase (AMPK) pathway signaling. In addition, the comparative transcriptome analysis revealed that zebrafish and mammals may share a common molecular response to GTE. Our findings suggest that daily consumption of green tea may be beneficial for the prevention and treatment of obesity.

An Integrative Evaluation Method for the Biological Safety of Down and Feather Materials.

BACKGROUND: Down and feather materials have been commonly used and promoted as natural stuffing for warm clothing and bedding. These materials tend to become more allergenic as they become contaminated with microorganisms, in addition to being subjected to several kinds of chemical treatments. The biological or chemical contaminants in these materials pose a major risk to human health, to consumers and manufacturers alike. Here, we report the development of an integrative evaluation method for down and feather materials to assess bacterial contamination and in vivo toxicity. METHODS: To assess bacterial contamination, we quantified 16S ribosomal RNA, performed culture tests, and established a conversion formula. To determine in vivo toxicity, we performed a zebrafish embryo toxicity testing (ZFET). RESULTS: Washing the material appropriately decreases the actual number of bacteria in the down and feather samples; in addition, after washing, 16S rRNA sequencing revealed that the bacterial compositions were similar to those in rinse water. The ZFET results showed that even materials with low bacterial contamination showed high toxicity or high teratogenicity, probably because of the presence of unknown chemical additives. CONCLUSIONS: We established an integrative evaluation method for down and feather safety, based on bacterial contamination with in vivo toxicity testing.

Novel immunologic tolerance of human cancer cell xenotransplants in zebrafish.

Immune deficiency or suppression in host animals is an essential precondition for the success of cancer cell xenotransplantation because the host immune system has a tendency to reject implanted cells. However, in such animals, the typical tumor microenvironment seen in cancer subjects does not form because of the lack of normal immunity. Here, we developed a novel zebrafish (Danio rerio) model based on 2 rounds of cancer cell xenotransplantation that achieved cancer-specific immunologic tolerance without immunosuppression. We irradiated human cancer cells (PC-3, K562 and HepG2) to abolish their proliferative abilities and implanted them into zebrafish larvae. These cells survived for 2 weeks in the developing host. Three months after the first implantation, the zebrafish were implanted with the same, but nonirradiated, cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. To reveal the transcriptional mechanism of this immune tolerance, we conducted dual RNA-seq of the tumor with its surrounding tissues and identified several regulatory zebrafish genes that are involved in immunity; the expression of plasminogen activator, urokinase, and forkhead box P3 was altered in response to immunologic tolerance. In conclusion, this xenograft method has potential as a platform for zebrafish-based anticancer drug discovery because it can closely mimic human clinical cancers without inducing immune suppression.

Copper Oxide Nanoparticles Reduce Vasculogenesis in Transgenic Zebrafish Through Down-Regulation of Vascular Endothelial Growth Factor Expression and Induction of Apoptosis.

The present study investigated the effects of exposure to metal oxide nanoparticles on vasculogenesis/angiogenesis using transgenic zebrafish. The study also examined the potential mechanisms involved in those effects using human umbilical vein endothelial cells (HUVEC). TG (nacre/fli1:EGFP) zebrafish were exposed to nano-sized titanium dioxide (TiO2), silica dioxide (SiO2), and copper oxide (CuO) particles at 0.01, 1 and 100 µg/ml concentrations from 1 to 5 dpf (day-post-fertilization). Angiogenesis was evaluated morphologically at the end of exposure. Exposure to CuO nanoparticles reduced the number of transversely-running subintestinal vessels in TG zebrafish. Exposure to CuO nanoparticles down-regulated the expression of vascular endothelial growth factor (VEGF) and VEGF receptor in endothelial cells sorted by Fluorescence Activated Cell Sorter (FACS). Exposure of HUVEC to CuO nanoparticles reduced cell viability and increased apoptotic index in a dose-dependent manner. The results suggested that CuO nanoparticles inhibit vasculogenesis through reduction of VEGF expression and induction of apoptosis.

In vivo selective imaging and inhibition of leukemia stem-like cells using the fluorescent carbocyanine derivative, DiOC5(3).

Elimination of leukemia stem cells (LSCs) is necessary for the destruction of malignant cell populations. Owing to the very small number of LSCs in leukemia cells, xenotransplantation studies are difficult in terms of functionally and pathophysiologically replicating clinical conditions of cell culture experiments. There is currently a limited number of lead compounds that target LSCs. Using the LSC-xenograft zebrafish screening method we previously developed, we found that the fluorescent compound 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) selectively marked LSCs and suppressed their proliferation in vivo and in vitro. DiOC5(3) had no obvious toxicity to human umbilical cord blood CD34+ progenitor cells and normal zebrafish. It accumulated in mitochondria through organic anion transporter polypeptides that are overexpressed in the plasma membrane of LSCs, and induced apoptosis via ROS overproduction. DiOC5(3) also inhibited the nuclear translocation of NF-κB through the downregulation of LSC-selective pathways, as indicated from DNA microarray analysis. In summary, DiOC5(3) is a new type of anti-LSC compound available for diagnostic imaging and therapeutics that has the advantage of being a single fluorescent chemical.

Downregulation of stanniocalcin 1 is responsible for sorafenib-induced cardiotoxicity.

Sorafenib is associated with adverse cardiac effects, including left ventricular dysfunction. However, the precise mechanism remains unclear. Here, we aimed to establish the genes responsible for this cardiotoxicity using zebrafish and human cardiomyocytes. Fluorescent cardiac imaging using pigmentless zebrafish with green fluorescent protein hearts revealed that the ventricular dimensions of the longitudinal axis with sorafenib were significantly shorter than those of the control group. Transcriptome analysis of their hearts revealed that stanniocalcin 1 (stc1) was downregulated by sorafenib. stc1 knockdown in zebrafish revealed that reduction of stc1 decreased the longitudinal dimensions of zebrafish ventricles, similar to that which occurs during sorafenib treatment. STC1 downregulation and cytotoxicity were also seen in human cardiomyocytes exposed to sorafenib. To clarify the molecular function of stc1 in sorafenib-induced cardiotoxicity, we focused on oxidative stress in cardiomyocytes treated with sorafenib. Reactive oxygen species (ROS) production significantly increased in both species of human cardiomyocytes and zebrafish exposed to sorafenib and STC1 knockdown compared with the controls. Finally, we found that forced expression of stc1 normalized impairment, decreasing the longitudinal dimensions in zebrafish treated with sorafenib. Our study demonstrated that STC1 plays a protective role against ventricular dysfunction and ROS overproduction, which are induced by sorafenib treatment. We discovered for the first time that STC1 downregulation is responsible for sorafenib-induced cardiotoxicity through activated ROS generation.

Zebrafish xenotransplantation model for cancer stem-like cell study and high-throughput screening of inhibitors.

Xenotransplantation studies are important tools for studying cancer biology, especially for assaying tumor cell malignancy and providing cancer information in vivo. Cancer stem-like cells (CSCs) have been identified in many cancer types to drive tumor growth and recurrence, from "keeping" to "keep" resistant to chemotherapy and radiation therapy. In this study, we developed the xenotransplantation of CSCs derived from the leukemia and solid tumor cell lines using the zebrafish models. In adult zebrafish, we investigated that the xenografted leukemia stem cells (LSCs) from K562 cells could proliferate in vivo and keep the cancer property by re-transplantation. As for the solid tumor, these CSCs from DU145 cells (human prostate cancer) and HepG2 cells (human liver cancer) could form the tumor mass and even metastasis after xenotransplantation. In addition, the zebrafish embryos with CSC xenotransplantation could evaluate docetaxel in vivo efficiently and be available to screen the novel inhibitors by high-throughput manner. In summary, these zebrafish xenotransplantation models devote a good platform for the CSC mechanism investigation and anti-CSC inhibitor screening.

Zinc finger MYND-type containing 8 promotes tumour angiogenesis via induction of vascular endothelial growth factor-A expression.

Zinc finger, MYND-type containing 8 (ZMYND8) encodes a receptor for activated C-kinase protein. Here, we report that ZMYND8 promotes angiogenesis in prostate cancer xenografts in zebrafish, as well as tube formation in human umbilical vascular endothelial cell (HUVEC) cultures. Using transcriptome analyses, we found upregulation of ZMYND8 expression in both zebrafish prostate cancer xenografts and prostate cancer samples from patients. In vitro and in vivo ZMYND8 knockdown suppressed angiogenesis, whereas ZMYND8 overexpression enhanced angiogenesis. Notably, ZMYND8 induced vegfa mRNA expression selectively in prostate cancer xenografts. Integrated analysis of human and zebrafish transcriptomes, which identified ZMYND8, might be a powerful strategy to determine also other molecular targets for inhibiting prostate cancer progression.

Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.

Lemon (Citrus limon) contains various bioactive flavonoids, and prevents obesity and obesity-associated metabolic diseases. We focused on eriocitrin (eriodictyol 7-rutinoside), a powerful antioxidative flavonoid in lemon with lipid-lowering effects in a rat model of high-fat diet. To investigate the mechanism of action of eriocitrin, we conducted feeding experiments on zebrafish with diet-induced obesity. Oral administration of eriocitrin (32 mg/kg/day for 28 days) improved dyslipidaemia and decreased lipid droplets in the liver. DNA microarray analysis revealed that eriocitrin increased mRNA of mitochondrial biogenesis genes, such as mitochondria transcription factor, nuclear respiratory factor 1, cytochrome c oxidase subunit 4, and ATP synthase. In HepG2 cells, eriocitrin also induced the corresponding orthologues, and reduced lipid accumulation under conditions of lipid loading. Eriocitrin increased mitochondrial size and mtDNA content, which resulted in ATP production in HepG2 cells and zebrafish. In summary, dietary eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.

Quantitative phenotyping-based in vivo chemical screening in a zebrafish model of leukemia stem cell xenotransplantation.

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.

Fluorescent-based methods for gene knockdown and functional cardiac imaging in zebrafish.

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.

Identification of a novel indoline derivative for in vivo fluorescent imaging of blood-brain barrier disruption in animal models.

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741.

Green tea extract suppresses adiposity and affects the expression of lipid metabolism genes in diet-induced obese zebrafish.

BACKGROUND: Visceral fat accumulation is one of the most important predictors of mortality in obese populations. Administration of green tea extract (GTE) can reduce body fat and reduce the risk of obesity-related diseases in mammals. In this study, we investigated the effects and mechanisms of GTE on adiposity in diet-induced obese (DIO) zebrafish. METHODS: Zebrafish at 3.5 to 4.5 months post-fertilization were allocated to four groups: non-DIO, DIO, DIO + 0.0025%GTE, and DIO + 0.0050%GTE. The non-DIO group was fed freshly hatched Artemia once daily (5 mg cysts/fish daily) for 40 days. Zebrafish in the three DIO groups were fed freshly hatched Artemia three times daily (60 mg cysts/fish daily). Zebrafish in the DIO + 0.0025%GTE and DIO + 0.0050%GTE groups were exposed to GTE after the start of feeding three times daily for 40 days. RESULTS: Three-dimensional microcomputed tomography analysis showed that GTE exposure significantly decreased the volume of visceral but not subcutaneous fat tissue in DIO zebrafish. GTE exposure increased hepatic expression of the lipid catabolism genes ACOX1 (acyl-coenzyme A oxidase 1, palmitoyl), ACADM (acyl-coenzyme A dehydrogenase, c-4 to c-12 straight chain), and PPARA (peroxisome proliferator-activated receptor alpha). GTE exposure also significantly decreased the visceral fat expression of SOCS3 (suppressor of cytokine signaling 3b) which inhibits leptin signaling. CONCLUSIONS: The present results are consistent with those seen in mammals treated with GTE, supporting the validity of studying the effects of GTE in DIO zebrafish. Our results suggest that GTE exerts beneficial effects on adiposity, possibly by altering the expression of lipid catabolism genes and SOCS3.

In vivo assessment of the permeability of the blood-brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish.

BACKGROUND: Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood-brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB. METHODS: We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7-8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs. RESULTS: The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs. CONCLUSIONS: We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.

Transcriptome analysis of anti-fatty liver action by Campari tomato using a zebrafish diet-induced obesity model.

BACKGROUND: High dietary intake of vegetable products is beneficial against obesity and its related diseases such as dyslipidemia, nonalcoholic fatty liver disease, and cancer. We previously developed a diet-induced obesity model of zebrafish (DIO-zebrafish) that develops visceral adiposity, dyslipidemia, and liver steatosis. Zebrafish is a polyphagous animal; thus we hypothesized that DIO-zebrafish could be used for transcriptome analysis of anti-obesity effects of vegetables. RESULTS: Each vegetable exhibited different effects against obesity. We focused on "Campari" tomato, which suppressed increase of body weight, plasma TG, and lipid droplets in livers of DIO-zebrafish. Campari tomato decreased srebf1 mRNA by increase of foxo1 gene expression, which may depend on high contents of ß-carotene in this strain. CONCLUSIONS: Campari tomato ameliorates diet-induced obesity, especially dyslipidemia and liver steatosis via downregulation of gene expression related to lipogenesis. DIO-zebrafish can discriminate the anti-obesity effects of different strains of vegetables, and will become a powerful tool to assess outcomes and find novel mechanisms of anti-obesity effects of natural products.

Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.

BACKGROUND: Obesity is a multifactorial disorder influenced by genetic and environmental factors. Animal models of obesity are required to help us understand the signaling pathways underlying this condition. Zebrafish possess many structural and functional similarities with humans and have been used to model various human diseases, including a genetic model of obesity. The purpose of this study was to establish a zebrafish model of diet-induced obesity (DIO). RESULTS: Zebrafish were assigned into two dietary groups. One group of zebrafish was overfed with Artemia (60 mg dry weight/day/fish), a living prey consisting of a relatively high amount of fat. The other group of zebrafish was fed with Artemia sufficient to meet their energy requirements (5 mg dry weight/day/fish). Zebrafish were fed under these dietary protocols for 8 weeks. The zebrafish overfed with Artemia exhibited increased body mass index, which was calculated by dividing the body weight by the square of the body length, hypertriglyceridemia and hepatosteatosis, unlike the control zebrafish. Calorie restriction for 2 weeks was applied to zebrafish after the 8-week overfeeding period. The increased body weight and plasma triglyceride level were improved by calorie restriction. We also performed comparative transcriptome analysis of visceral adipose tissue from DIO zebrafish, DIO rats, DIO mice and obese humans. This analysis revealed that obese zebrafish and mammals share common pathophysiological pathways related to the coagulation cascade and lipid metabolism. Furthermore, several regulators were identified in zebrafish and mammals, including APOH, IL-6 and IL-1ß in the coagulation cascade, and SREBF1, PPARα/γ, NR1H3 and LEP in lipid metabolism. CONCLUSION: We established a zebrafish model of DIO that shared common pathophysiological pathways with mammalian obesity. The DIO zebrafish can be used to identify putative pharmacological targets and to test novel drugs for the treatment of human obesity.

In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives.

BACKGROUND: The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae. RESULTS: We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1µg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells. CONCLUSIONS: The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.