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Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators...
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... more recent findings indicate that dorsomorphin may have additional targets, we also analyzed a more selective BMP inhibitor, LDN-193189 [28]. As shown in Table 2, pulsing with 70 µM pCAME resulted in 6.7% ectopic tails. Pulsing with 5 or 10 µM LDN-193189 did not yield any tail duplications. ...
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... In our continuing search for new bioactive compounds from cold-water habitats, an extract of the tunicate Synoicum sp. collected in Antarctica was screened in a phenotypic zebrafish assay [37]. This bioassay utilizes the rapid and ex vivo development of zebrafish embryos to enable monitoring of phenotypic changes caused by extracts, fractions, and compounds of interest. ...
... Synoicum sp. extract-treated embryos exhibited truncation of the trunk and tail and overall developmental delay, indicating the potential involvement of multiple signaling pathways known to be important for embryonic development and anterior-posterior axis specification [37], therefore prioritizing this extract for further investigation. In this paper we report the isolation of four new indolone alkaloids, australindolones A-D (1-4), as well as the isolation of a new indole alkaloid, meridianin H (12), and the previously reported meridianins A-G (5-11) ( Figure 1). ...
... Complicating the discussion, at the concentration tested, these rotations are near the limit of detection of the polarimeter. However, small rotations, including several with rotations under 10 degrees (absolute value) have been reported for 3-substituted oxindolones [37]. Whether the australindolones are racemic or scalemic remains to be determined. ...
Five new alkaloids have been isolated from the lipophilic extract of the Antarctic tunicate Synoicum sp. Deep-sea specimens of Synoicum sp. were collected during a 2011 cruise of the R/V Nathanial B. Palmer to the southern Scotia Arc, Antarctica. Crude extracts from the invertebrates obtained during the cruise were screened in a zebrafish-based phenotypic assay. The Synoicum sp. extract induced embryonic dysmorphology characterized by axis truncation, leading to the isolation of aminopyrimidine substituted indolone (1–4) and indole (5–12) alkaloids. While the primary bioactivity tracked with previously reported meridianins A–G (5–11), further investigation resulted in the isolation and characterization of australindolones A–D (1–4) and the previously unreported meridianin H (12).
... A stable mutant zebrafish line corresponds better to the situation in human patients with genomic mutations. However, an "acute" genomic KO/mutant approach allows for fast screening early on, and easily produces scalable numbers of KO larvae for large scale experiments such as chemical screens [8,9]. ...
Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.
... He et al., 2009a; He et al., 2009b; Hong et al., 2009; He et al., 2010;Alex et al., 2010;KRILL et al., 2010;Zhu et al., 2011;Liu et al., 2011;Crawford et al., 2011; Tse et al., 2012;Zhong et al., 2012;He et al., 2012;Zhang et al., 2012;Han et al., 2012;Yu et al., 2013; Ba et al., 2013;Lee et al., 2013; Yahg et al., 2013; Fan et al., 2013; Yue et al., 2013, Da-Song et al., 2014; Zhou et al., 2014; Germano et al., 2014 Lai et al., 2015 Antithrombotic activity Song et al., 2012; Shi et al., 2015 anticonvulsant Orellana-Paucar et al., 2012; Buenafe et al., 2013; Challal et al., 2014 Anti-inflammatory Kao et al., 2010; Bohni et al., 2013; Wang et al., 2013 Antilipemic Jin et al., 2011; Kim et al., 2012; Pardal et al., 2014; Littleton et al., 2014 MelanogenicKim et al., 2008;Ding et al., 2011;Park et al., 2013;Kim et al., 2014;Wang et al., 2014 Neurodegenerative diseases Zhang 2012Chong et al., 2013;Guo et al., 2014 Toxicity Gertch et al., 2003 Kelleher et al., 2009;Wang et al., 2011; Fang et al., 2011;Zhang et al., 2014;Ding and Chen, 2012; Bernadi et al., 2013 Others studiesWang et al., 2009; Del Valle-Mojica and Ortiz, 2012; Wei et al., 2012a,b;Ferri-Lagneau et al., 2012;Gebruers et al., 2013;Yu et al., 2013;Ulloa et al., 2013;Kang et al., 2014;Holbech et al., 2013 ...
Research on natural products is facing significant difficulty. The analysis "high-throughput screening" has limited effectiveness in their evaluation. This report on in vitro screening of pharmacological activity of the drugs candidate molecule by evaluating a single target. The presence of many substances in a natural extract makes this process unprecise. The zebrafish (Danio rerio, ZF) is well suited to high-throughput applications owing to its high fecundity, rapid extrauterine development and transparency during organogenesis. This fact promotes increase in the relevance of their use in biological assays, which is hard to be matched in vitro. Tests based on ZF can aid in the isolation of bioactive molecules from plant extracts, which was identified in a large-scale screening. This increases the biological relevance of such findings. Another decisive factor for the use of ZF as an experimental model, for widespread testing, is the few extracts or the isolated compounds required for tests. Therefore, the growing number of publications and innovative models created for the research shows a lot of diseases with this species, revealing the importance of ZF as an experimental model for the screening of natural products.
... The loss of BMP signaling during late gastrulation stages of zebrafish development results in the formation of a single ectopic tail on the ventral side of the primary tail (Connors et al., 1999;Pyati et al., 2005). The ectopic tail frequently contains notochord cells (Gebruers et al., 2013;Pyati et al., 2005;Yang and Thorpe, 2011). We reasoned that this might represent a splitting of the dorsal and ventral MPC populations, which would provide further evidence for the existence of the two MPC populations that we identified by genetic and lineage tracing-based methods. ...
... In embryos with ectopic tails, the hypochord extends into the ectopic tail (along with some notochord), and is completely absent from the midline in the primary tail in regions posterior to the ectopic tail (Fig. 6F,F′,H-H″). By contrast, floor plate is continuous in the primary tail anterior and posterior to the ectopic tail (Fig. S1), and previous work has demonstrated that neural tissue, including floor plate, is never found in the ectopic tail (Gebruers et al., 2013;Pyati et al., 2005;Yang and Thorpe, 2011). These results indicate that embryos with ectopic tails undergo a separation of the dorsal and ventral MPC populations, and that ventral MPC derivatives (notochord and hypochord) populate the ectopic tail and dorsal MPC derivatives (notochord and floor plate) reside in the primary tail. ...
... In zebrafish, loss of BMP signaling during late gastrulation or early somitogenesis stages results in a partially penetrant phenotype of an ectopic tail on the ventral side of the posterior embryo (Pyati et al., 2005;Stickney et al., 2007;Yang and Thorpe, 2011). The same phenotype occurs after loss of non-canonical Wnt signaling, and in both cases the posterior notochord is bifurcated along the dorsalventral axis, with one part in the normal notochord domain and the other extending into the ectopic ventral tail (Gebruers et al., 2013;Pyati et al., 2005;Yang and Thorpe, 2011). This phenotype arises from defective cell migration (Yang and Thorpe, 2011). ...
Vertebrate body axis formation depends on a population of bipotential neuromesodermal cells along the posterior wall of the tailbud that make a germ layer decision after gastrulation to form spinal cord and mesoderm. Despite exhibiting germ layer plasticity, these cells never give rise to midline tissues of the notochord, floor plate and dorsal endoderm, raising the question of whether midline tissues also arise from basal posterior progenitors after gastrulation. We show in zebrafish that local posterior signals specify germ layer fate in two basal tailbud midline progenitor populations. Wnt signaling induces notochord within a population of notochord/floor plate bipotential cells through negative transcriptional regulation of sox2. Notch signaling, required for hypochord induction during gastrulation, continues to act in the tailbud to specify hypochord from a notochord/hypochord bipotential cell population. Our results lend strong support to a continuous allocation model of midline tissue formation in zebrafish, and provide an embryological basis for zebrafish and mouse bifurcated notochord phenotypes as well as the rare human congenital split notochord syndrome. We demonstrate developmental equivalency between the tailbud progenitor cell populations. Midline progenitors can be transfated from notochord to somite fate after gastrulation by ectopic expression of msgn1, a master regulator of paraxial mesoderm fate, or if transplanted into the bipotential progenitors that normally give rise to somites. Our results indicate that the entire non-epidermal posterior body is derived from discrete, basal tailbud cell populations. These cells remain receptive to extracellular cues after gastrulation and continue to make basic germ layer decisions.
... Two main fields were prevalent among EDA studies using zebrafish bioassays: drug discovery from natural products and environmental toxicology ( Figure 3). Natural product studies aimed to identify bioactive compounds for pharmacological applications, investigating mostly plant extracts [19,[34][35][36][37][38][39][40][41][42][43][44][45] but also extracts of bacteria [46], cyanobacteria and algae [47], seaweed [48] and marine organisms [49]. Environmental toxicology studies aimed to identify the toxic compounds in various environmental samples, including marine and fluvial sediments [50][51][52], soil [53], cyanobacteria and algae [54,55], industrial effluent [33], rubber tyre leachates [32], oil sand process waters [56,57] and river pore water [58]. ...
... dimethyl sulfoxide (DMSO), in concentrations ranging from 0.01% [35], 0.1% [31,45], 0.2% [26], 0.5% [46,47], 1% [33,[48][49][50] up to 2% [51]. In addition, ethanol was also used as a carrier by a few studies, in concentrations of 0.001% for experiments with larvae [40,41] or 0.435% for experiments with zebrafish adults [42]. ...
... Assessment of teratogenesis and developmental effects was done in studies that identified the bioactive compounds from microalgae, cyanobacteria and plant [41,54,55], river pore water [58] and developmental toxicants in soil [53]. Most studies evaluated traditionally assessed morphological endpoints, while one investigation of plant fractions focused on ectopic tail formation [41]. ...
Bioassays play a central role in effect-directed analysis (EDA), and their selection and application have to consider rather specific aspects of this approach. Meanwhile, bioassays with zebrafish, an established model organism in different research areas, are increasingly being utilized in EDA. Aiming to contribute for the optimal application of zebrafish bioassays in EDA, this review provides a critical overview of previous EDA investigations that applied zebrafish bioassays, discusses the potential contribution of such methods for EDA and proposes strategies to improve future studies. Over the last 10 years, zebrafish bioassays have guided EDA of natural products and environmental samples. The great majority of studies performed bioassays with embryos and early larvae, which allowed small-scale and low-volume experimental setups, minimized sample use and reduced workload. Biotesting strategies applied zebrafish bioassays as either the only method guiding EDA or instead integrated into multiple bioassay approaches. Furthermore, tiered biotesting applied zebrafish methods in both screening phase as well as for further investigations. For dosing, most of the studies performed solvent exchange of extracts and fractions to dimethyl sulfoxide (DMSO) as carrier. However, high DMSO concentrations were required for the testing of complex matrix extracts, indicating that future studies might benefit from the evaluation of alternative carrier solvents or passive dosing. Surprisingly, only a few studies reported the evaluation of process blanks, indicating a need to improve and standardize methods for blank preparation and biotesting. Regarding evaluated endpoints, while acute toxicity brought limited information, the assessment of specific endpoints was of strong value for bioactivity identification. Therefore, the bioassay specificity and sensitivity to identify the investigated bioactivity are important criteria in EDA. Additionally, it might be necessary to characterize the most adequate exposure windows and assessment setups for bioactivity identification. Finally, a great advantage of zebrafish bioassays in EDA of environmental samples is the availability of mechanism- and endpoint-specific methods for the identification of important classes of contaminants. The evaluation of mechanism-specific endpoints in EDA is considered to be a promising strategy to facilitate the integration of EDA into weight-of-evidence approaches, ultimately contributing for the identification of environmental contaminants causing bioassay and ecological effects.
... Fish and amphibians provide efficient models to identify chemicals that alter biological processes (Tal et al., 2010;Tomlinson et al., 2012, Gebruers et al., 2013. Their embryos and larvae can be reared in microtiter plates to rapidly screen well-characterized or novel compounds for effects at molecular, cell, developmental, or whole-organism levels. ...
... However, because of the sparsity of data, we expect that this observation may not truly reflect the reality. In particular, orthology-based target predictions are likely to be useful in species like zebrafish, as attested by many biological studies that map small molecule activity across vertebrate species (Gebruers et al., 2013;Ridges et al., 2012). ...
Motivation: The functional impact of small molecules is increasingly being assessed in different eukaryotic species through large-scale phenotypic screening initiatives. Identifying the targets of these molecules is crucial to mechanistically understand their function and uncover new therapeutically relevant modes of action. However, despite extensive work carried out in model organisms and human, it is still unclear to what extent one can use information obtained in one species to make predictions in other species.
Results: Here, for the first time, we explore and validate at a large scale the use of protein homology relationships to predict the targets of small molecules across different species. Our results show that exploiting target homology can significantly improve the predictions, especially for molecules experimentally tested in other species. Interestingly, when considering separately orthology and paralogy relationships, we observe that mapping small molecule interactions among orthologs improves prediction accuracy, while including paralogs does not improve and even sometimes worsens the prediction accuracy. Overall, our results provide a novel approach to integrate chemical screening results across multiple species and highlight the promises and remaining challenges of using protein homology for small molecule target identification.
Availability and implementation: Homology-based predictions can be tested on our website http://www.swisstargetprediction.ch.
Contact:david.gfeller@unil.ch or vincent.zoete@isb-sib.ch.
Supplementary information:Supplementary data are available at Bioinformatics online.
... Zebrafish chemical screens have also been used to identify novel active components in naturally occurring substances. A bioactivity was discovered in an extract from the Jasminum gilgianum plant using a zebrafish screen [46]. This extract was found to induce the formation of ectopic tailbuds in larvae. ...
... Once again, candidate pathways, in this case BMP and non-canonical Wnt signaling, were tested using established knowledge of zebrafish developmental biology. When an inhibitor of JNK, an important kinase in the Wnt noncanonical planar cell polarity (PCP) pathway was tested on ectopic tailbud formation, the inhibitor (SP600125) was found to phenocopy pCAME [46]. In addition, strong synergy was observed when pCAME was combined with BMP inhibitor dorsomorphin (also identified in a zebrafish chemical screen [47]) providing further evidence that the new compound modulates non-canonical Wnt signaling. ...
In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies.
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... To illustrate, Yu et al. (2008) screened for the dorsalized phenotype characterized by curved/woundup/undeveloped tail to search for inhibitors of the Bone Morphogenetic Protein (BMP) pathway, which led to the discovery of dorsomorphin [46]. A more recent study by Gebruers et al. (2013) reports the discovery of a possible pathway-targeting agent, para-coumaric acid methyl ester isolated from Jasminum gilgianum, which induced an ectopic tail phenotype, suggesting a role as a modifier of either the BMP or non-canonical Wnt pathway [47]. ...
... To illustrate, Yu et al. (2008) screened for the dorsalized phenotype characterized by curved/woundup/undeveloped tail to search for inhibitors of the Bone Morphogenetic Protein (BMP) pathway, which led to the discovery of dorsomorphin [46]. A more recent study by Gebruers et al. (2013) reports the discovery of a possible pathway-targeting agent, para-coumaric acid methyl ester isolated from Jasminum gilgianum, which induced an ectopic tail phenotype, suggesting a role as a modifier of either the BMP or non-canonical Wnt pathway [47]. ...
Targeted therapy, the treatment of cancer based on an underlying genetic alteration, is rapidly gaining favor as the preferred therapeutic approach. To date, although natural products represent a rich resource of bio-diverse drug candidates, only a few have been identified to be effective as targeted cancer therapies largely due to the incompatibilities to current high-throughput screening methods. In this article, we review the utility of a zebrafish developmental screen for bioactive natural product-based compounds that target signaling pathways that are intimately shared with those in humans. Any bioactive compound perturbing signaling pathways identified from phenotypic developmental defects in zebrafish embryos provide an opportunity for developing targeted therapies for human cancers. This model provides a promising tool in the search for targeted cancer therapeutics from natural products.
... Most recently, the development of genome-editing techniques, including transcription activator-like effector nucleases (TALENs) (Joung and Sander, 2013) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Chang et al., 2013; Hruscha et al., 2013; Hwang et al., 2013), has enabled researchers to induce heritable mutations with remarkable precision (see poster panel 2). Finally, large-scale screens using panels of pharmaceutical compounds or small molecules have been developed over the past several years, where thousands of embryos can be allocated into multi-well plates and exposed to chemicals to test drug dose-response levels, potential for teratogenicity or toxicity, and therapeutic potential of known or novel compounds (Peterson et al., 2000; Brannen et al., 2013; Ducharme et al., 2013; Esterberg et al., 2013; Gebruers et al., 2013; Hao et al., 2013; Swartz et al., 2013; Wang et al., 2014; Westhoff et al., 2013) (see poster panel 2). ...
Advances in genomics and next-generation sequencing have provided clinical researchers with unprecedented opportunities to understand the molecular basis of human genetic disorders. This abundance of information places new requirements on traditional disease models, which have the potential to be used to confirm newly identified pathogenic mutations and test the efficacy of emerging therapies. The unique attributes of zebrafish are being increasingly leveraged to create functional disease models, facilitate drug discovery, and provide critical scientific bases for the development of new clinical tools for the diagnosis and treatment of human disease. In this short review and the accompanying poster, we highlight a few illustrative examples of the applications of the zebrafish model to the study of human health and disease.
