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). 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