Laboratory Course Using Zebrafish to Uncover Changing Roles of Wnt Signaling in Early Vertebrate Development.

Coordinated signaling pathway activity directs early patterning to set up the vertebrate body plan. Perturbations in the timing or location of signal molecule expression impacts embryo morphology and organ formation. In this study, we present a laboratory course to use zebrafish for studying the role of Wnt signaling in specifying the early embryonic axes. Students are exposed to basic techniques in molecular and developmental biology, including embryo manipulation, fluorescence microscopy, image processing, and data analysis. Furthermore, this course incorporates student-designed experiments to stimulate independent inquiry and improve scientific learning, providing an experience resembling graduate-level laboratory research. Students appreciated following vertebrate development in real-time, and principles of embryogenesis were reinforced by observing the morphological changes that arise due to signaling alterations. Scientific and research skills were enhanced through practice in experimental design, interpretation, and presentation.

Transcriptomic profile in carbendazim-induced developmental defects in zebrafish (Danio rerio) embryos/larvae.

Carbendazim is a widely used fungicide to protect agricultural and horticultural crops against a wide array of fungal species. Published reports have shown that the wide usage of carbendazim resulted in reprotoxicity, carcinogenicity, immunotoxicity, and developmental toxicity in mammalian models. However, studies related to the developmental toxicity of carbendazim in aquatic organisms are not clear. To address this gap, an attempt was made by exposing zebrafish embryos to carbendazim (800 µg/L) and assessing the phenotypic and transcriptomic profile at different developmental stages [24 hour post fertilization (hpf), 48 hpf, 72 hpf and 96 hpf). At 48 hpf, phenotypic abnormalities such as delay in hatching rate, deformed spinal axial curvature, and pericardial edema were observed in zebrafish larvae over its respective controls. At 72 hpf, exposure of zebrafish embryos exposed to carbendazim resulted in scoliosis; however, unexposed larvae did not exhibit signs of scoliosis. Interestingly, the transcriptomic analysis revealed a total of 1253 DEGs were observed at selected time points, while unique genes at 24 hpf, 48 hpf, 72 hpf and 96 hpf was found to be 76.54 %, 61.14 %, 92.98 %, and 68.28 %, respectively. Functional profiling of downregulated genes revealed altered transcriptomic markers associated with phototransduction (24 hpf and 72 hpf), immune system (48 hpf), and SNARE interactions in the vesicular pathway (96 hpf). Whereas functional profiling of upregulated genes revealed altered transcriptomic markers associated with riboflavin metabolism (24 hpf), basal transcription factors (48 hpf), insulin signaling pathway (72 hpf), and primary bile acid biosynthesis (96 hpf). Taken together, carbendazim-induced developmental toxicity could be ascribed to pleiotropic responses at the molecular level, which in turn might reflect phenotypic abnormalities.

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A. franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A. franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A. franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A. franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A. franciscana, an organism of practical importance as a feed source in aquaculture.

Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

Assessments of carbon nanotubes toxicities in zebrafish larvae using multiple physiological and molecular endpoints.

In recent years, carbon nanotubes (CNTs) have become one of the most promising materials for the technology industry. However, due to the extensive usage of these materials, they may be released into the environment, and cause toxicities to the organism. Here, their acute toxicities in zebrafish embryos and larvae were evaluated by using various assessments that may provide us with a novel perspective on their effects on aquatic animals. Before conducting the toxicity assessments, the CNTs were characterized as multiwall carbon nanotubes (MWCNTs) functionalized with hydroxyl and carboxyl groups, which improved their solubility and dispersibility. Based on the results, abnormalities in zebrafish behaviors were observed in the exposed groups, indicated by a reduction in tail coiling frequency and alterations in the locomotion as the response toward photo and vibration stimuli that might be due to the disruption in the neuromodulatory system and the formation of reactive oxygen species (ROS) by MWCNTs. Next, based on the respiratory rate assay, exposed larvae consumed more oxygen, which may be due to the injuries in the larval gill by the MWCNTs. Finally, even though no irregularity was observed in the exposed larval cardiac rhythm, abnormalities were shown in their cardiac physiology and blood flow with significant downregulation in several cardiac development-related gene expressions. To sum up, although the following studies are necessary to understand the exact mechanism of their toxicity, the current study demonstrated the environmental implications of MWCNTs in particularly low concentrations and short-term exposure, especially to aquatic organisms.

Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos.

Tobacco pollutants are prevalent in the environment, leading to inadvertent exposure of pregnant females. Studies of these pollutants' toxic effects on embryonic development have not fully elucidated the potential underlying mechanisms. Therefore, in this study, we aimed to investigate the developmental toxicity induced by cigarette smoke extract (CSE) at concentrations of 0.25, 1, and 2.5% using a zebrafish embryo toxicity test and integrated transcriptomic analysis of microRNA (miRNA) and messenger RNA (mRNA). The findings revealed that CSE caused developmental toxicity, including increased mortality and decreased incubation rate, in a dose-dependent manner. Moreover, CSE induced malformations and apoptosis, specifically in the head and heart of zebrafish larvae. We used mRNA and miRNA sequencing analyses to compare changes in the expression of genes and miRNAs in zebrafish larvae. The bioinformatics analysis indicates that the mechanism underlying CSE-induced developmental toxicity was associated with compromised genetic material damage repair, deregulated apoptosis, and disturbed lipid metabolism. The enrichment analysis and RT-qPCR show that the ctsba gene plays a crucial function in embryo developmental apoptosis, and the fads2 gene mainly regulates lipid metabolic toxicity. The results of this study improve the understanding of CSE-induced developmental toxicity in zebrafish embryos and contribute insights into the formulation of novel preventive strategies against tobacco pollutants during early embryonic development.

Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.

Hexaconazole induces developmental toxicities via apoptosis, inflammation, and alterations of Akt and MAPK signaling cascades.

Hexaconazole is a highly effective triazole fungicide that is frequently applied in various countries to elevate crop productivity. Given its long half-life and high water solubility, this fungicide is frequently detected in the environment, including water sources. Moreover, hexaconazole exerts hazardous effects on nontarget organisms. However, little is known about the toxic effects of hexaconazole on animal development. Thus, this study aimed to investigate the developmental toxicity of hexaconazole to zebrafish, a valuable animal model for toxicological studies, and elucidate the underlying mechanisms. Results showed that hexaconazole affected the viability and hatching rate of zebrafish at 96 h postfertilization. Hexaconazole-treated zebrafish showed phenotypic defects, such as reduced size of head and eyes and enlarged pericardiac edema. Moreover, hexaconazole induced apoptosis, DNA fragmentation, and inflammation in developing zebrafish. Various organ defects, including neurotoxicity, cardiovascular toxicity, and hepatotoxicity, were observed in transgenic zebrafish models olig2:dsRed, fli1:eGFP, and l-fabp:dsRed. Furthermore, hexaconazole treatment altered the Akt and MAPK signaling pathways, which possibly triggered the organ defects and other toxic mechanisms. This study demonstrated the developmental toxicity of hexaconazole to zebrafish and elucidated the underlying mechanisms.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Iprodione induces hepatotoxicity in zebrafish by mediating ROS generation and upregulating p53 signalling pathway.

Iprodione is an effective and broad-spectrum fungicide commonly used for early disease control in fruit trees and vegetables. Due to rainfall, iprodione often finds its way into water bodies, posing toxicity risks to non-target organisms and potentially entering the human food chain. However, there is limited information available regarding the developmental toxicity of iprodione specifically on the liver in existing literature. In this study, we employed larval and adult zebrafish as models to investigate the toxicity of iprodione. Our findings revealed that iprodione exposure led to yolk sac edema and increased mortality in zebrafish. Notably, iprodione exhibited specific effects on zebrafish liver development. Additionally, zebrafish exposed to iprodione experienced an overload of reactive oxygen species, resulting in the upregulation of p53 gene expression. This, in turn, triggered hepatocyte apoptosis and disrupted carbohydrate/lipid metabolism as well as energy demand systems. These results demonstrated the substantial impact of iprodione on zebrafish liver development and function. Furthermore, the application of astaxanthin (an antioxidant) and p53 morpholino partially mitigated the liver toxicity caused by iprodione. To summarize, iprodione induces apoptosis through the upregulation of p53 mediated by oxidative stress signals, leading to liver toxicity in zebrafish. Our study highlights that exposure to iprodione can result in hepatotoxicity in zebrafish, and it may potentially pose toxicity risks to other aquatic organisms and even humans.

DDT exposure induces tremor-like behavior and neurotoxicity in developmental stages of embryonic zebrafish.

Dichlorodiphenyltrichloroethane (DDT) is a broad-spectrum insecticide, widely detected in environments due to its high stability characteristic and long natural half-life period. The adverse impact of DDT exposure on organisms and humans has attracted great concern worldwide. The current study explored the developmental and neurobehavioral toxicity response of DDT in embryonic zebrafish. The embryos were treated with DDT (0, 0.1, 1, 2.5 and 5 µM) during 6 h post fertilization (hpf) to 144 hpf. Our result indicated that DDT exposures increased the embryo hatching rate at 48 and 60 hpf, the larval malformation rate at 120 hpf and mortality rate at 144 hpf. The manifested malformations included uninflated swim bladder, bent spine and tail, deformed liver, and pericardial edema. The 120 hpf larval organs size of the gut and swim bladder was decreased in higher exposed concentration groups. Besides, DDT exposure resulted in hyperactivity for the embryo spontaneous movement at 24 hpf and tremor like movement measured by the free larval activity at 72 hpf, as well as the larval activity at 96 hpf under light-dark transition stimulus. Mechanistic examinations at 120 hpf revealed DDT exposure elevated oxidative stress through MDA formation increase, ATP level decrease as well as antioxidant enzyme genes (sod1 and gpx1a) expression decrease. DDT exposure induced abnormal neurotransmitters expression with DA level increase, 5-HT and NOS level decrease. DDT exposure suppressed the gene expressions involved in axon development (rab33a and nrxn2a) and potassium channel (kcnq2 and kcnq3). Our results suggest that the hyperactivity and tremor like movement in DDT-exposed embryos/larvae may result from oxidative stress involved with neuronal damage.

A mechanical wave travels along a genetic guide to drive the formation of an epithelial furrow during Drosophila gastrulation.

Epithelial furrowing is a fundamental morphogenetic process during gastrulation, neurulation, and body shaping. A furrow often results from a fold that propagates along a line. How fold formation and propagation are controlled and driven is poorly understood. To shed light on this, we study the formation of the cephalic furrow, a fold that runs along the embryo dorsal-ventral axis during Drosophila gastrulation and the developmental role of which is still unknown. We provide evidence of its function and show that epithelial furrowing is initiated by a group of cells. This cellular cluster works as a pacemaker, triggering a bidirectional morphogenetic wave powered by actomyosin contractions and sustained by de novo medial apex-to-apex cell adhesion. The pacemaker's Cartesian position is under the crossed control of the anterior-posterior and dorsal-ventral gene patterning systems. Thus, furrow formation is driven by a mechanical trigger wave that travels under the control of a multidimensional genetic guide.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

Investigation of ammonia-induced lethal toxicity toward ion regulation in zebrafish embryos.

Ammonia is an environmental pollutant that is toxic to all aquatic animals. However, the mechanism of ammonia toxicity toward the ion regulatory function of early-stage fish has not been fully documented. We addressed this issue using zebrafish embryos as a model. We hypothesized that ammonia might impair ion regulation by inducing oxidative stress, mitochondrial dysfunction, and cell death of epidermal ionocytes and keratinocytes in zebrafish embryos. After exposure to various concentrations (10- 30 mM) of NH4Cl for 96 h, mortality increased up to 50 % and 100 % at 25 and 30 mM, respectively. Whole-embryo sodium, potassium, and calcium contents decreased at ≥10 mM, suggesting dysfunction of ion regulation. Numbers of H+-ATPase-rich (HR) cells and Na+/K+-ATPase-rich (NaR) cells (two ionocyte subtypes) were not significantly altered at 15 or 20 mM, while the mitochondrial abundance significantly decreased and reactive oxygen species (ROS) levels significantly increased in ionocytes. Moreover, caspase-3-dependent apoptosis was found in epidermal keratinocytes. Whole-embryo transcript levels of several genes involved in ion regulation, antioxidation, and apoptosis were upregulated after ammonia exposure. In conclusion, ammonia exposure was shown to induce oxidative stress and mitochondrial dysfunction in ionocytes and apoptosis in keratinocytes, thereby impairing ion regulation and ultimately leading to the death of zebrafish embryos.

PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in Caenorhabditis elegans embryos.

The kinase PAR-4/LKB1 is a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use Caenorhabditis elegans to examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a previously unappreciated function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation, but rather to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which both restricts intestinal specification to the E lineage and ensures its robust differentiation.

Targeted design of synthetic enhancers for selected tissues in the Drosophila embryo.

Enhancers control gene expression and have crucial roles in development and homeostasis1-3. However, the targeted de novo design of enhancers with tissue-specific activities has remained challenging. Here we combine deep learning and transfer learning to design tissue-specific enhancers for five tissues in the Drosophila melanogaster embryo: the central nervous system, epidermis, gut, muscle and brain. We first train convolutional neural networks using genome-wide single-cell assay for transposase-accessible chromatin with sequencing (ATAC-seq) datasets and then fine-tune the convolutional neural networks with smaller-scale data from in vivo enhancer activity assays, yielding models with 13% to 76% positive predictive value according to cross-validation. We designed and experimentally assessed 40 synthetic enhancers (8 per tissue) in vivo, of which 31 (78%) were active and 27 (68%) functioned in the target tissue (100% for central nervous system and muscle). The strategy of combining genome-wide and small-scale functional datasets by transfer learning is generally applicable and should enable the design of tissue-, cell type- and cell state-specific enhancers in any system.

N-nitrosodimethylamine exposure to zebrafish embryos/larvae causes cardiac and spinal developmental toxicity.

N-nitrosodimethylamine (NDMA), one of the new nitrogen-containing disinfection by-products, is potentially cytotoxic, genotoxic, and carcinogenic. Its potential toxicological effects have attracted a wide range of attention, but the mechanism is still not sufficiently understood. To better understand the toxicological mechanisms of NDMA, zebrafish embryos were exposed to NDMA from 3 h post-fertilization (hpf) to 120hpf. Mortality and malformation were significantly increased, and hatching rate, heart rate, and swimming behavior were decreased in the exposure groups. The result indicated that NDMA exposure causes cardiac and spinal developmental toxicity. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, and bcl-2 were significantly affected by NDMA exposure. Moreover, the genes associated with spinal and cardiac development (myh6, myh7, nkx2.5, eph, bmp2b, bmp4, bmp9, run2a, and run2b) were significantly downregulated after treatment with NDMA. Wnt and TGF-ß signaling pathways, crucial for the development of diverse tissues and organs in the embryo and the establishment of the larval spine, were also significantly disturbed by NDMA treatment. In summary, the disinfection by-product, NDMA, exhibits spinal and cardiac developmental toxicity in zebrafish embryos, providing helpful information for comprehensive analyses and a better understanding the mechanism of its toxicity.

Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S).

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73-0.90; R2FET48h = 0.79-0.90; R2FET96h = 0.76-0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70-0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68-0.74 and R2FET96h = 0.62-0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) - 90.9% (FET test) using ZFL and in 30.7% (AFT test) - 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.