Zebrafish Assay Research Articles

Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays

Zebrafish, during embryo, larvae and adult stages were selected to investigate the potential environmental risk and aquatic toxicity of a widely used fungicide, difenoconazole. In addition to mortality, embryo development endpoints, teratogenic effects and behavior abnormity were measured. Finally, the developmental parameters of the adult fish were assessed after 14 days' exposure. This study concluded that the acute toxicity of difenoconazole to the three phases of zebrafish were larvae (1.17 mg/L) > adult fish (1.45 mg/L) > embryo (2.34 mg/L). A large suite of symptoms was induced in embryonic development by different dosages of difenoconazole, including hatching inhibition, abnormal spontaneous movement, slow heart rate, growth regression and morphological deformities. 0.50 mg/L of difenoconazole could cause significant body color blackening and decrease in the heart rate of zebrafish larvae over 24 h. In addition, 0.25 mg/L of difenoconazole apparently inhibited the growth weight of adult zebrafish measured after 14 days' exposure.

Eaf1 and Eaf2 negatively regulate canonical Wnt/β-catenin signaling

Eaf factors play a crucial role in tumor suppression and embryogenesis. To investigate the potential mechanism of Eaf activity, we performed loss- and gain-of-function assays in zebrafish using morpholino and mRNA injections, respectively. We found that eaf1 and eaf2 inhibit Wnt/β-catenin signaling, thereby modulating mesodermal and neural patterning in the embryo. Moreover, ectopic expression of eaf1 and eaf2 in embryos and cultured cells blocked β-catenin reporter activity. By immunoprecipitation, we also observed that Eaf1 and Eaf2 bound to the Armadillo repeat region and C-terminus of β-catenin, as well as to other β-catenin transcription complex proteins, such as c-Jun, Tcf and Axin, suggesting the formation of a novel complex. In addition, the N-terminus of Eaf1 and Eaf2 bound to β-catenin and exhibited dominant-negative activity, whereas the C-terminus appeared to either harbor a suppression domain or to recruit a repressor. Both the N- and C-terminus must be intact for Eaf1 and Eaf2 suppressive activity. Lastly, we demonstrate a conservation of biological activities for Eaf family proteins across species. In summary, our evidence points to a novel role for Eaf1 and Eaf2 in inhibiting canonical Wnt/β-catenin signaling, which might form the mechanistic basis for Eaf1 and Eaf2 tumor suppressor activity.

14-Membered Macrocyclic Ring-Derived Toolbox: The Identification of Small Molecule Inhibitors of Angiogenesis and Early Embryo Development in Zebrafish Assay

A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respectively. 1.4c was identified as an antiangiogenesis agent.

Macrocyclic Glycohybrid Toolbox Identifies Novel Antiangiogenesis Agents from Zebrafish Assay

A practical and modular approach to obtain a diverse set of 14-membered macrocyclic compounds from carbohydrates is developed that utilizes functional groups at C-1 and C-5. The evaluation of this toolbox in various zebrafish assays led to the identification of 2.7f as an antiangiogenesis agent.

Zebrafish Assays as Developmental Toxicity Indicators in The Design of TAML Oxidation Catalysts.

TAML activators promise a novel water treatment approach by efficiently catalysing peroxide-based degradation of chemicals of high concern at environmental concentrations. Green design ethics demands an exploration of TAML toxicity. Exposure to high concentrations of certain activators caused adverse effects in zebrafish. At typical TAML operational concentrations, development was not perturbed.

Tetrahymena: An Alternative Model Host for Evaluating Virulence of Aeromonas Strains

An easier assessment model would be helpful for high-throughput screening of Aeromonas virulence. The previous study indicated the potential of Tetrahymena as a permissive model to examine virulence of Aeromonas hydrophila. Here our aim was to assess virulence of Aeromonas spp. using two model hosts, a zebrafish assay and Tetrahymena-Aeromonas co-culture, and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model. First, virulence of 39 Aeromonas strains was assessed by determining the 50% lethal dose (LD50) in zebrafish. LD50 values ranging from 1.3×102 to 3.0×107 indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model. In Tetrahymena-Aeromonas co-culture, we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena. An Aeromonas isolate was considered virulent when its relative survival was greater than 60%, while the Aeromonas isolate was considered avirulent if its relative survival was below 40%. When relative survival of T. thermophila was lower than 40% after co-culture with an Aeromonas isolate, the bacterial strain was regarded as virulent. In contrast, the strain was classified as avirulent if relative survival of T. thermophila was greater than 50%. Encouragingly, data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models. The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

Evaluation of the Anti-angiogenic Activity of Saponins from Maesa lanceolata by Different Assays

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts. In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Multiple enhancers associated with ACAN suggest highly redundant transcriptional regulation in cartilage

The chondroitin sulfate proteoglycan core protein aggrecan is the major protein constituent of cartilage aside from collagen, and is largely responsible for its distinctive mechanical properties. Aggrecan is required both for proper cartilage formation in development and maintenance of mature cartilage. Prominent ACAN transcription is a conserved feature of vertebrate cartilage, although little is known about its specific transcriptional regulation. We examined the genomic interval containing human ACAN for transcriptional enhancers directing expression to cartilage, using a functional assay in transgenic zebrafish. We tested 24 conserved non‐coding sequences, representing ~6% of the total sequence in the interval, and identified eleven independently capable of regulating reporter gene expression in cartilage. These enhancers were widely spaced, from >100kb upstream of the gene to within the first intron. While the majority displayed broad cartilage expression in zebrafish larvae, several were restricted to a subset of cartilage cells in the craniofacial skeleton. In older fish, the enhancers displayed differential activity; some maintained expression, either in all cartilage or preferentially in articular cartilage at the joints, while others were not active. This remarkable degree of overlapping regulatory control has been highly conserved; we identified clear orthologues of six enhancers at the chicken ACAN locus, arranged in the same order relative to the gene. These were also functional in directing expression to cartilage in transgenic zebrafish. Several enhancers contain potential binding sites for Sox9, consistent with its described role as an upstream regulator of ACAN expression. However, others lacked Sox9 consensus binding sites, implicating additional pathways and transcription factors as regulators of ACAN expression in cartilage, either in development or adult tissue. Our identification of these enhancer sequences is the necessary first step in detailed examination of the upstream regulators of ACAN expression.

Transcriptional Enhancers in Protein-Coding Exons of Vertebrate Developmental Genes

Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77%) exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less). GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%), suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147) or tissue-specificity (coding: 14/24 vs. noncoding: 50/105). Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81). Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

Exons Encode Enhancers

Genetics Transcription factor binding to enhancer elements is critical for proper gene regulation. Enhancers are often found in noncoding sequences in close proximity to the gene that they regulate and sometimes even on another chromosome; however, whether they are also found in exons, the coding regions of DNA, is unclear. Birnbaum et al. analyzed 25 mouse and human enhancer-associated ChIP-seq data sets in order to identify enhancer peaks that overlap exons and found regulatory transcription factor binding to exonic regions. In fact, in mice, roughly 7% of enhancer peaks overlapped coding exons. Mutation of these elements in zebrafish and mouse enhancer assays showed that although exonic sequences are necessary, they are not sufficient for full enhancer function. Absence of an exon-encoded enhancer, however, did have functional consequences. Thus, exonic sequences may function in the regulation of nearby genes. Moreover, phenotypes seen in genetic knockout animals may be the result of not only the lack of expression of the deleted gene but also alterations in the expression of genes that are regulated by enhancers in the deleted exons. Genome Res. 22 , 10.1101/gr.133546.111 (2012).

Lens development depends on a pair of highly conserved Sox21 regulatory elements

Highly conserved non-coding elements (CNEs) linked to genes involved in embryonic development have been hypothesised to correspond to cis-regulatory modules due to their ability to induce tissue-specific expression patterns. However, attempts to prove their requirement for normal development or for the correct expression of the genes they are associated with have yielded conflicting results. Here, we show that CNEs at the vertebrate Sox21 locus are crucial for Sox21 expression in the embryonic lens and that loss of Sox21 function interferes with normal lens development. Using different expression assays in zebrafish we find that two CNEs linked to Sox21 in all vertebrates contain lens enhancers and that their removal from a reporter BAC abolishes lens expression. Furthermore inhibition of Sox21 function after the injection of a sox21b morpholino into zebrafish leads to defects in lens development. These findings identify a direct link between sequence conservation and genomic function of regulatory sequences. In addition to this we provide evidence that putative Sox binding sites in one of the CNEs are essential for induction of lens expression as well as enhancer function in the CNS. Our results show that CNEs identified in pufferfish-mammal whole-genome comparisons are crucial developmental enhancers and hence essential components of gene regulatory networks underlying vertebrate embryogenesis.

Ototoxin-induced cellular damage in neuromasts disrupts lateral line function in larval zebrafish

The ototoxicity of a number of marketed drugs is well documented, and there is an absence of convenient techniques to identify and eliminate this unwanted effect at a pre-clinical stage. We have assessed the validity of the larval zebrafish, or more specifically its lateral line neuromast hair cells, as a microplate-scale in vivo surrogate model of mammalian inner ear hair cell responses to ototoxin exposure. Here we describe an investigation of the pathological and functional consequences of hair cell loss in lateral line neuromasts of larval zebrafish after exposure to a range of well known human and non-human mammalian ototoxins. Using a previously described histological assay, we show that hair cell damage occurs in a concentration-dependent fashion following exposure to representatives from a range of drug classes, including the aminoglycoside antibiotics, salicylates and platinum-based chemotherapeutics, as well as a heavy metal. Furthermore, we detail the optimisation of a semi-automated method to analyse the stereotypical startle response in larval zebrafish, and use this to assess the impact of hair cell damage on hearing function in these animals. Functional assessment revealed robust and significant attenuation of the innate startle, rheotactic and avoidance responses of 5 day old zebrafish larvae after treatment with a number of compounds previously shown to induce hair cell damage and loss. Interestingly, a startle reflex (albeit reduced) was still present even after the apparent complete loss of lateral line hair cell fluorescence, suggesting some involvement of the inner ear as well as the lateral line neuromast hair cells in this reflex response. Collectively, these data provide evidence to support the use of the zebrafish as a pre-clinical indicator of drug-induced histological and functional ototoxicity.

On the edge: Pharmacological evidence for anxiety-related behavior in zebrafish larvae

Zebrafish larvae are ideally suited for high-throughput analyses of vertebrate behavior. The larvae can be examined in multiwell plates and display a range of behaviors during early development. Previous studies have shown that zebrafish larvae display a preference for the edge of the well and several lines of evidence suggest this edge preference (thigmotaxis) may be a measure of anxiety. In the present study, we further examined the relation between edge preference and anxiety by imaging zebrafish larvae exposed to three psychoactive drugs diazepam (Valium), fluoxetine (Prozac), and caffeine. The edge preference was first examined in a five-fish assay, with and without visual stimuli. Diazepam, a benzodiazepine that binds to GABA receptors, reduced the larval edge preference, with or without visual stimuli. In contrast, fluoxetine, a selective serotonin reuptake inhibitor, did not affect the edge preference. Caffeine increased the preference for the edge in response to visual stimuli. Similar effects were observed in a two-fish assay; diazepam-exposed larvae showed a reduced edge preference and caffeine exposed larvae showed an increased edge preference. These results suggest that the edge preference in zebrafish larvae is a measure of anxiety and further illustrate that the pharmaceuticals used in the study have different mechanisms of action. Although there are substantial differences between zebrafish and human brains, our results indicate that the signals that regulate anxiety are similar on a molecular level. We propose that high-throughput assays in zebrafish may be used to uncover genetic or environmental factors that cause anxiety disorders and may contribute to the development of novel strategies to prevent or treat such disorders.

Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4-dichlorophenol

Chlorophenols in the aquatic environment have been of concern due to their potential effects on human and wildlife. In the present study, the endocrine disrupting effects of 2,4-dichlorophenol (2,4-DCP) were investigated in vitro and in vivo. In the in vitro assay, H295R human adrenocortical carcinoma cells were used to determine the potential effects of 2,4-DCP on steroidogenesis. Exposure to 0, 0.1, 0.3 or 1.0 mg 2,4-DCP/L resulted in less production of 17β-estradiol (E2) and alterations in transcript expressions of genes involved in steroidogenesis, including cytochrome P450 ( CYP11A, CYP17, CYP19), 3βHSD, 17βHSD and StAR. In the in vivo study, effects of 0, 0.03, 0.1 or 0.3 mg 2,4-DCP/L on concentrations of steroid hormones in plasma of adult zebrafish ( Danio rerio) were measured and expression of mRNA of selected genes in hypothalamic-pituitary-gonadal (HPG) axis and liver were determined. Exposure of zebrafish to 2,4-DCP resulted in lesser concentrations of E2 accompanied by down-regulation of CYP19A mRNA in the females. In males, exposure to 2,4-DCP resulted in greater concentrations of testosterone (T) and E2 along with greater mRNA expression of CYP17 and CYP19A. The mRNA expression of prostaglandin synthase ( Ptgs2) gene, which regulates ovulation, was down-regulated in females, but up-regulated in males. The hepatic estrogenic receptor ( ERα and ERβ) and vitellogenin ( VTG1 and VTG3) mRNAs were up-regulated in both females and males. The average number of eggs spawned was significantly less upon exposure to 2,4-DCP. Exposure of adult zebrafish to 2,4-DCP resulted in lesser rates of hatching of eggs. The results demonstrated that 2,4-DCP modulates transcription of steroidogenetic genes in both H295R cells and in the zebrafish HPG-axis and disrupts steroidogenesis, which in turn, can cause adverse effects on reproduction in fish.

Genome-Wide Programming of Myeloid Cell Fates

Abstract SCI-29Hematopoiesis represents one of the most tractable models of adult stem cell development and differentiation. Transcription factor (TF) proteins have long been recognized as major regulators of blood stem cell development as well as the subsequent differentiation into the multiple mature hematopoietic lineages. Seminal studies in multiple vertebrate model systems have identified specific TFs that control cell fate choices during myeloid differentiation (1). It remains largely unknown, however, how individual TFs are integrated into wider transcriptional regulatory networks, and how combinatorial TF interactions within these networks drive lineage specific gene expression programs. We are addressing these issues using two complementary approaches. First, we use a combination of transgenic reporter assays and network modeling approaches to reconstruct core transcription factor networks operating in early myeloid differentiation. Second, we employ genome-scale analysis of transcription factor binding sites for key hematopoietic regulators in both stem/progenitor cells and mature lineages (2,3). Integrated analysis of genome-scale datasets reveals previously unrecognized combinatorial interactions within core hematopoietic regulatory networks, which can be validated using both biochemical and mouse knockout approaches. Moreover, our studies also pinpoint novel candidate hematopoietic regulators, several of which we have validated using high throughput loss-of-function assays in zebrafish. Disclosures:No relevant conflicts of interest to declare.

Assessment of lateral line function: A potential technique for studies in ototoxicity

In zebrafish neuromast hair cells, the process of programmed cell death ototoxic damage is strikingly similar to that of degenerating hair cells of the mammalian organ of Corti. Therefore, in vivo zebrafish assays involving the lateral line have been developed for drug ototoxicity screening. This is accomplished by examination of canal neuromast morphology in treated larvae using fluorescent dyes. To-date however, physiological confirmation of lateral line dysfunction resulting from such ototoxins has not been reported in the scientific literature – neither for larval nor adult zebrafish. Here we describe a rapid, non-invasive far-field electrophysiological method for assessing lateral line function. We suggest that ototoxic and otoprotective agents identified in larval studies may be assessed using this tool in adult fish. In this way, potential drug candidates can be further screened en route to testing in mammalian models, before potential clinical trials begin.

A high‐content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling

Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF.