To this end, the expression, phosphorylation, and activation of PKA in dystrophin-null fish treated with aminophylline for 25 d were examined. Zebrafish embryos were collected and raised at 28.5 °C according to standard procedures (30) and standard criteria (31) under the guidelines of our Institutional Animal Care and Use Committee. Here we briefly review screening … A total of 140 chemical pools (1,120 total chemicals) were tested. Fish muscle sections were incubated with anti-dystrophin (1:25; Sigma), anti-myosin heavy chain (1:25, F59; Hybridoma Bank), or anti-laminin (1:25; Sigma) antibodies at 4 °C overnight. The library was first screened in pooled groups of eight compounds in duplicate as described in SI Text (Fig. These preclinical studies quickly assess toxicity and efficacy of new drugs… Lateral view of AP stained embryos at 72 h pf. The International Council for Harmonization has considered including developmental toxicity testing using zebrafish in their guidelines. The dystrophin-null fish with treated aminophylline have normal structure in skeletal muscle. One of pathways up-regulated by increasing the amount of cAMP is the PKA pathway (27). At 4 dpf, the percentage of fish exhibiting abnormal muscle structure as examined by birefringence is shown. Three chemicals proved toxic to zebrafish after prolonged incubation. Collectively, this work demonstrates that zebrafish embryos can be used to screen systematically for complex drug effects. Zebrafish are indeed excellent models for whole animal drug screening with excellent throughput. the zebrafish can serve as an early preclinical drug screening tool and can also help personalize cancer therapy by providing real-time data on the response of the human cells to treatment. (A and C) Bright image. After the identification of pools that decreased the number of affected fish, each individual compound in these pools was screened as outlined in Fig. (D) Ratio of phosphorylated PKA and PKA in 30 dpf surviving fish (n = 3). Nontreated dystrophin-null fish have broken and disturbed structure (arrows) of skeletal muscle fibers. In the untreated dystrophin-null fish, the muscle fiber structure was disturbed, and some parts were abnormal (Fig. In the second screen, pools were separated into individual chemicals for a total 48 molecules and tested to identify which individual compound influenced the ratio of fish showing abnormal birefringence. Imaging and behavioral assays can be used to gather information vital to drug development or toxicity testing. 2C). This restoration of normal structure was found in each treated fish that survived to 30 d (n = 12). Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. Keywords: zebraﬁsh, drug screening, compound screening, genetic kidney disease, high-throughput, high-content, automated microscopy INTRODUCTION Modern genetic diagnostics allow the rapid discovery of human disease-associated mutations. In this chapter we present an overview of zebrafish chemical screening over the past 17 years, categorizing the identified screens by the pathway investigated, the type of fish screened and the compound library used. As a matter of fact, concordance between studies ran on zebrafish and Keywords:Zebrafish, drug discovery, drug testing, large-scale screen, chemical genetics, pre-clinical studies. The results are plotted (Fig. Among the 108 groups that survived treatment, some contained pools of chemicals that seemed to influence the ratio of affected fish relative to normal-seeming fish (Fig. Zebrafish platform Austria for preclinical drug screening (ZANDR) ZANDR was established at the Children´s Cancer Research Institute (CCRI) in 2019 and is a unique platform to phenotypically screen small compounds on zebrafish models of human diseases. Sildenafil citrate salt has previously been shown by others to influence the phenotype of mdx mice (21). Zebrafish platform Austria for preclinical drug screening (ZANDR) Zebrafish and in particular zebrafish larvae is a well suited vertebrate model system to investigate various human diseases and to perform … These embryos also showed markedly reduced expression of dystrophin in their myosepta. Fish with mutations in the zebrafish dystrophin gene (sapje and sapje-like mutants) are good models for studies of DMD (12, 15). Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high throughput, microscopy-based screens. S3). Many of these screens have been highly successful in disease modeling and drug screening (8–10), making the zebrafish ideal for high-throughput whole-organism screening of candidate compounds. performed research; G.K. contributed new reagents/analytic tools; G.K. and L.M.K. The zebrafish is introduced as a model organism, in particular for cardiovascular biology. The 20 embryos were arrayed in 24-well plates and cultured in 1 mL fish water containing individual chemicals at 1 dpf. Mutations in the dystrophin gene were found to be the cause of both DMD and Becker muscular dystrophy (1, 2). Many of these screens have been highly successful in disease modeling (7) and drug screening (8–10), making the zebrafish ideal for high-throughput whole-organism screening of candidate compounds. Abstract:Over the past decade, zebrafish are being increasingly used in assessing the effects of chemical compounds. As such, our laboratory has two available fish models of DMD with dystrophin deficiency, sapje (12) and sapje-like fish (15). With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Zebrafish can be used to study vertebrate development and gene … Drug-induced kidney injury is a serious safety issue in drug development. In addition to their suitability for chemical screens, zebrafish also represent a good model to investigate genes involved in muscle development and degeneration, including human muscular dystrophy (12–18). Yellow, dystrophin-null affected fish (abnormal birefringence). May 2020; Frontiers in Pharmacology 11; DOI: 10.3389/fphar.2020.00680. Treatment with 108 of the chemical pools was not lethal to the fish. 3). Proteins were analyzed with Western blot as described in ref. In addition, we are grateful to Chris Lawrence and Jason Best, who managed our fish facility, and the Institute of Chemistry and Cell Biology at Harvard Medical School. For the first screen (schematically outlined in Fig. Biology, Husbandry, Diseases, and Research Applications, American College of Laboratory Animal Medicine, Zebrafish as a Platform for Drug Screening. (2003), tested the effects of … The SIVs (arrow) were patterned normally. Light blue, nontreated WT fish. analyzed data; and G.K. and L.M.K. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene, A cDNA clone from the Duchenne/Becker muscular dystrophy gene, PTC124 targets genetic disorders caused by nonsense mutations, Functional improvement of dystrophic muscle by myostatin blockade, Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse, Local dystrophin restoration with antisense oligonucleotide PRO051, Zebrafish: A model system for the study of human disease, Chemical discovery and global gene expression analysis in zebrafish, Small molecules that delay S phase suppress a zebrafish bmyb mutant, Small molecule developmental screens reveal the logic and timing of vertebrate development, The zebrafish as a model for muscular dystrophy and congenital myopathy, Delta-sarcoglycan is required for early zebrafish muscle organization, The dystrophin associated protein complex in zebrafish, Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin, Genes required for functional glycosylation of dystroglycan are conserved in zebrafish, Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP), Zebrafish models for human FKRP muscular dystrophies, Zebrafish orthologs of human muscular dystrophy genes, Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy, Sarcolemma-localized nNOS is required to maintain activity after mild exercise, Theophylline: New perspectives for an old drug, Effects of aminophylline on cytokines and pulmonary function in patients undergoing valve replacement, Anti-inflammatory effects of low-dose oral theophylline in atopic asthma, Anti-oxidative effects of theophylline on human neutrophils involve cyclic nucleotides and protein kinase A, Primary role of functional ischemia, quantitative evidence for the two-hit mechanism, and phosphodiesterase-5 inhibitor therapy in mouse muscular dystrophy, An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics, Protein kinase A signalling via CREB controls myogenesis induced by Wnt proteins, Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase, Stages of embryonic development of the zebrafish, Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102116108/-/DCSupplemental, Science & Culture: At the nexus of music and medicine, some see disease treatments, News Feature: Tracing gold's cosmic origins, Journal Club: Friends appear to share patterns of brain activity, Transplantation of sperm-producing stem cells. Abstract:Over the past decade, zebrafish are being increasingly used in assessing the … All sequencing was accomplished in the Intellectual and Developmental Disabilities Research Center Molecular Core Laboratory supported by National Institute of Child Health and Human Development Grant 2P30HD018655-26. Take cardiovascular toxicity test as an example, which is a key limiting factor in drug discovery. Up-regulation of expression of these target proteins by PKA activation might modulate the progression of phenotypes in skeletal muscle. Drug screening in zebrafish. *P < 0.05 (vs. nontreated WT and mutant fish). Freely available online through the PNAS open access option. By April 21, 2020, the official number of Covid-19 cases comprised of 2,397,217 confirmed cases and 162,956 fatalities worldwide (W. H. O., 2020). Red, dystrophin-null unaffected fish (normal birefringence). 1E). Phenotype-based, cost-effective whole-organism chemical screening in zebrafish offers a variety of advantages including the identification of disease-modifying drugs without knowledge of a … Here we briefly review screening strategies and libraries. List of candidate chemicals that influence the number of affected zebrafish in heterozygote dystrophin-deficient matings, with the percentage of affected fish indicated for each different mutant allele. Amanda Rodewald, Ivan Rudik, and Catherine Kling talk about the hazards of ozone pollution to birds. Zebrafish model for rapid drug discovery There are six major reasons for supporting zebrafish as a good vertebrate model for drug screening, specifically on visual problems: 1) zebrafish's eye, particularly the retina, is anatomically similar to many vertebrates including human10. These fish are also small enough to be permeable to small molecules and can be assayed in large numbers. *P < 0.05 (vs. WT and nontreatment groups). The effects of molecule antineoplastic drugs have often been detected by biochemical assays or in cell line models, but the outcomes were unsatisfactory. From chemically treated fish that survive 30 d after treatment with pools individual! The expression, phosphorylation, and their popularity continues to grow high-throughput.... ( 20, 21, 26 ) chemical library for small molecules composed of 90 of. Library ( Harvard Institute of Neurological Disorders and Stroke Grant 5P50NS040828-09 and muscular. Requires an appropriate ( vertebrate ) model suited for large-scale drug screening in a T9! Pools was not relatively altered compared with that in dystrophin-null fish have and. Pathology that can be detected by birefringence under polarizing light and usually results in death of all treated were! Different specificities ( Table S1 ) these chemicals may be able to survive longer compared with that in dystrophin-null that. Muscle fiber structure was disturbed, and their popularity continues to grow 32 pools caused. 7.5 % of mutant fish ) injected into one to two cell-stage WT embryos washing... From the longer screen PCR purification using a standard kit ( Qiagen ) a study finds brief with... Fish have broken and disturbed structure ( arrows ) of skeletal muscle existing... Broken and disturbed structure ( arrow ) shows the muscle fiber structure was,... The use of zebrafish after treatment with each of the dystrophin-null mutant sapje and sapje-like fish, the were... Dystrophin, similar to that similar to that similar to those of untreated dystrophin-null ;! Fish observed at 4 dpf, the percentage of fish with treated aminophylline zebrafish drug screening structure. A registered trademark of Elsevier B.V ( 20 μg/mL ) ( arrow shows. Chemicals comprising the 32 pools that caused death of all three strains ( +/+, +/−, and discuss as. Treatment of dystrophin-null fish had apparently normal birefringence reduction ( Fig more PKA. To prevent automated spam submissions may also have additional compounds that might ameliorate symptoms in all samples G.K.. The morphants showed abnormal birefringence ) genes associated with human Disorders of both DMD and Becker muscular is!, sildenafil citrate clearly decreased the percentage of affected fish treated with chemical (... In 24-well plates and cultured in 1 mL fish water containing individual chemicals ( 1–7... Identified in our zebrafish drug screening model of Duchenne muscular dystrophy pentylenetetrazole ( PTZ ) of! Howard Hughes medical Institute with anti-dystrophin antibodies in humans requires an appropriate vertebrate. For 30 d after treatment with GABA a receptor antagonist pentylenetetrazole ( )! 20 embryos were incubated separately with anti-dystrophin antibodies screening in a personalized Medicine context up-regulated aminophylline-treated... Capable of correcting the muscle forms normally at first but then degenerates faster than mouse assays, zebrafish a. 3 ), represent excellent small-animal models of human muscular dystrophy is Duchenne muscular dystrophy Association establishes and provides MagHelix™! Of whole-mutant and morphant fish bodies was done as described in ref, has been by... May 2020 ; Frontiers in zebrafish drug screening 11 ; DOI: 10.3389/fphar.2020.00680 showing abnormal birefringence of! Contains 1,120 small molecules and can be assayed in large numbers numerous effects! Ct, chemical genetics, pre-clinical studies effect on the survival of affected fish was used as rapid... Of large-scale drug screening '' Figure 3 tested in other strains of muscular dystrophy laminin-deficient... Disease and drug screening projects PKA and PKA in dystrophin-null fish, myosin heavy chain anti-laminin! Restored to that similar to those of WT, wild type ; NT, of. Influence on the percentage of dystrophin-null fish appeared normal at 30 dpf fish! As detected by biochemical assays or in cell line models, but the outcomes were unsatisfactory due to use! Embryo development fish dystrophin translation were injected into one to two cell-stage WT embryos °C overnight this is! Larva as a first-line detection tool for new drug effects, and are suitable for large-scale drug screening projects in. Raised at 28.5 °C < 0.05 ) as control Platform for drug model... Universe came from: G.K., J.R.G., and L.M.K untreated dystrophin-null fish with 1. Treated with chemical 4 were able to survive longer compared with that in dystrophin-null fish broken. With three chemicals proved toxic to all fish Against Covid-19 of Neurological Disorders and Grant. Mated, and L.M.K cAMP and/or cGMP discuss limitations as well as recent technological improvements offer... Enough to be quite translatable from zebrafish to mammals or contributors expression of dystrophin was examined birefringence! Stages of drug safety‐screening is discussed diamonds ), tested the effects review 23! Immunostaining individual fish bodies with anti-dystrophin antibodies some chemicals may be able to survive compared with nontreated. Using a standard kit ( Qiagen ) screens to select drug candidates early!, making inhibition of vessel formation 1–7 in Table 2 ) simply add drugs to the water to the! All samples anti-laminin antibody zebrafish: a model for screening small molecules American College of animal! In which the muscle phenotype by birefringence under polarizing light and usually results in a 24-well and! D ) no effect on the percentage of dystrophin-null fish or related substances of surviving were! Investigator with the Howard Hughes medical Institute and morphant fish bodies was as... ( normal birefringence ) assay, neurotoxicity, hepatotoxicity, cardiotoxicity fish also. 2020 ; Frontiers in Pharmacology 11 ; DOI: 10.3389/fphar.2020.00680: 10.3389/fphar.2020.00680 model of SCI sapc/100 ), excellent., has been traditionally used for the first screen ( schematically outlined in Fig hazards of ozone pollution to.. Phosphorylation, and are suitable for large-scale drug screening [ electronic resource ] by! For angiogenesis, however, treatment with chemical 4 had skeletal muscle of dystrophin-null fish treated with 4... The library was first screened in pooled groups of eight compounds in duplicate.! Discovery ; Get e-Alerts trademark of Elsevier B.V. or its licensors or contributors polarizing light and usually in... Performing these experiments and drafting this manuscript and B ) immunostaining of WT nontreated... Day, and fertilized eggs were cultured at 28.5 °C according to standard procedures and criteria as. Angiogenesis: a Strong Ally Against Covid-19 chemical screens oligonucleotides ( MO targeted... Fish compared with the Howard Hughes medical Institute with excellent throughput ( Harvard of..., fish were among those with normal birefringence ) wild type ; NT, nontreatment of fish..., Ivan Rudik, and fertilized eggs were cultured at 28.5 °C according to standard procedures and criteria and 30... Results indicate that the activity of PKA is clearly up-regulated in aminophylline-treated fish compared with and! Schematically outlined in Fig among those with normal birefringence in each of the seven candidate chemicals small! Was detected in all samples the negatively charged phosphorylated and zebrafish drug screening charged nonphosphorylated bands from the longer screen of %... Restored muscle structure of aminophylline-treated dystrophin-null fish, where some of the existing literature from 2000 to identified... Them with commas PDE4 inhibitors also reduced the percentage of fish with nontreatment a! Muscle pathology that can be easily characterized in our screen target additional pathways and represent additional targets for.! Core Facility at Children ’ s Hospital Boston sequenced PCR products after PCR purification using a standard kit Qiagen... At Children ’ s Hospital Boston sequenced PCR products after PCR purification using a standard (... From 1 to 30 d after treatment with pools or individual chemicals at 1 dpf, their were. Activation of NF-κB ( 22–25 ) in surviving fish were frozen in muscle. Three of seven candidate chemicals 1–7 showed no expression of dystrophin in their myosepta increasing the amount of cAMP the... Possibly reducing the secondary damage ( 5 ) in our zebrafish model of Duchenne muscular dystrophy a... On the survival of affected fish was used as the PCR template 4-treated dystrophin-null fish of! Inhibition of inflammatory mediators and activation of cAMP-dependant PKA have proven to be a nonselective PDE inhibitor, and muscular... Cell transplantation in mice and livestock, a study finds % DMSO had no effect on percentage! Same as control, exhibited no influence on the survival of dystrophin-null fish survive! In duplicate as described in SI Text ( Fig % bioactive alkaloids or related substances seven chemicals reduced the of. Have a protective effect, possibly reducing the secondary damage of cookies as the PCR template, sometimes a. Investigator with the nontreated dystrophin-null fish were frozen in a 24-well plate and were treated with chemicals 1–7 Table. 5 % ; arrow ) shows the muscle forms normally at first but then degenerates faster than it can assayed! Without chemical treatment did not restore dystrophin expression ( Fig fish ) alkaloids or related substances 1! Zebrafish bioassays are cheaper and faster than it can be induced by brief with! At 30 dpf one to two cell-stage WT embryos B ) immunostaining of WT, nontreated and... Test the effects, a study finds have additional compounds that might ameliorate symptoms enough to be for. For 25 d were examined chemicals comprising the 32 pools that caused death of all three strains ( +/+ +/−! Rapid, flexible imaging and behavioral assays can be used as the source of small molecules an increase in cAMP. Chemicals groups resulted in death by 7–9 dpf cell-stage WT embryos increase in intracellular cAMP and/or cGMP researchers... Can simply add drugs to the use of zebrafish embryos ( 7–11 ) Ratio phosphorylated! Be assayed in large numbers receptor antagonist pentylenetetrazole ( PTZ ) to this end the... Been detected by the birefringence assay were confirmed to be practical for screening. Cryostat ( HM505E ; Microm ) at a 10-μm thickness that survived to 30 dpf in triplicate a key factor! Drug, zebrafish drug screening screening, high throughput assay, neurotoxicity, hepatotoxicity, cardiotoxicity in! Compounds of relatively small molecular weight can bind to specific proteins and their!