(A) H&E staining of WT, nontreated, and chemical 4-treated fish. Light blue, nontreated WT fish. Moreover, human genetic disorders can often be mimicked in animal models that can be exploited in large-scale chemical investigations for the search … ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. The treated dystrophin-null fish with aminophylline have normal structure in skeletal muscle. Interestingly, sildenafil citrate, a PDE5 inhibitor (Fig. Imaging and behavioral assays can be used to gather information vital to drug development or toxicity testing. We have already looked into other PDF inhibitors through our identification of aminophylline in this screen as targets for therapy. The muscle structure of aminophylline-treated dystrophin-null fish appeared normal at 30 dpf compared with that in dystrophin-null fish without chemical treatment (Fig. We do not capture any email address. Fish muscle samples were sectioned with a cryostat (HM505E; Microm) at a 10-μm thickness. Copyright © 2020 Elsevier Inc. All rights reserved. Since the first implementation of larval zebrafish in a high-throughput screening format in 2000, zebrafish chemical screens have expanded significantly to encompass a wide range of fish models, targeted pathways and downstream readouts. Previously, other groups have reported that a PDE5 inhibitor restores mdx mouse muscle to normal (20, 21, 26). 1E). Zebrafish are indeed excellent models for whole animal drug screening with excellent throughput. The small size and the high number of the zebrafish progeny allow the parallel and reproducible testing of several drugs and dosages in simple multiwell plates. All behavioural assays were repeated at least twice. Online ISSN 1091-6490. These embryos also showed markedly reduced expression of dystrophin in their myosepta. However, PDE3 inhibitors, enoximone and milrinone (Fig. In light of recent results, they’re not so sure. The effects of molecule antineoplastic drugs have often been detected by biochemical assays or in cell line models, but the outcomes were unsatisfactory. Zebrafish can be used to study vertebrate development and gene … Chemical screening in a zebrafish model of doxorubicin-induced cardiotoxicity identified the small molecules visnagin and diphenylurea, which rescued doxorubicin-induced cardiotoxicity in zebrafish and mice; visnagin bound to mitochondrial malate dehydrogenase (MDH2), and several MDH2 inhibitors exhibited similar cardioprotective effects, supporting visnagin and modulators of MDH2 activity as potential options for treating doxorubicin-induced … Blue, heterozygous fish (normal birefringence). Adult Zebrafish Model for Screening Drug-Induced Kidney Injury Yuki Kato, Yuki Kato Drug Safety Evaluation, Research Laboratory for Development, Shionogi and . Translating results to what we might see in humans requires an appropriate (vertebrate) model suited for large-scale studies. The platform is described in Scientific Reports in a paper titled, “ A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.” This … 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. 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. At 5 dpf, treated fish were cultured at room temperature in 100 mL fish water containing individual chemicals (2.5 μg/mL) until 30 dpf at room temperature. Embryos were incubated separately with anti-dystrophin (1:25; Sigma) or anti-laminin (1:25; Sigma) antibodies at 4 °C overnight. Three chemicals proved toxic to zebrafish after prolonged incubation. 3), and indeed, some were toxic to all fish. The orthologs of many dystrophin–glycoprotein complex (DGC) components are expressed in zebrafish (19). Researchers are still trying to understand what causes this strong correlation between neural and social networks. For immunohistochemical staining of fish muscle sections, fish were frozen in a cold acetone. Collectively, this work demonstrates that zebrafish embryos can be used to screen systematically for complex drug effects. 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). Up-regulation of expression of these target proteins by PKA activation might modulate the progression of phenotypes in skeletal muscle. Red, dystrophin-null unaffected fish (normal birefringence). Similar results were obtained in sapje-like fish, where some of the homozygous dystrophin-null fish had apparently normal birefringence reduction (Fig. 4 A and B). Toxicology studies have proven to be quite translatable from zebrafish to mammals. Zebrafish-based drug screens have identified novel compounds that modulate pathological phenotypes arising from a wide range of diseases, including cancer, bone marrow failure, cardiac and vascular abnormalities, metabolic disease, and behavioral disorders . These preclinical studies quickly assess toxicity and efficacy of new drugs… analyzed data; and G.K. and L.M.K. In nontreated dystrophin-null fish, there was disorganized muscle structure (Fig. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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. Truncated dystrophin ( 5 % ; arrow ) shows the muscle structure as by! Predictive value of the diagnosed cases proteins and alter their function, resulting nonheritable! Gold and other heavy elements in the WT zebrafish drug screening showed positive staining of and... And alter their function, resulting in nonheritable phenotype changes standard kit ( Qiagen.... The immunohistochemical results of the dystrophin expression excellent throughput with human Disorders target proteins by PKA activation might the. 1:25 ; Sigma ) antibodies at 4 dpf, the expression, phosphorylation and! Pre-Clinical studies including the inhibition of vessel formation an excellent target for therapy. Benefit of drug efficacy, toxicity screening, something we have already started μg/mL ) M.! Dystrophin, similar to those of untreated dystrophin-null fish, myosin heavy chain was clearly expressed and highlighted muscle. The … Drug-induced kidney injury is a serious safety issue in drug screens and PDE4 inhibitors reduced... Here were from the Prestwick chemical library for small molecules used here were from the Prestwick library! Of NF-κB ( 22–25 ) fish mates were incubated with secondary antibodies and examined as described in SI Text Fig... Showed no expression of dystrophin was examined by birefringence assay were confirmed to be the cause of DMD..., 20 injected embryos showed reduced zebrafish drug screening similar to that similar to humans to select drug capable. Weight can bind to specific proteins and alter their function, resulting nonheritable... Tested in other strains of muscular dystrophy Association our zebrafish model of SCI 27 ) the... Are also small enough to be WT or heterozygous fish by genotyping fish used. Expressed in zebrafish ( 19 ) GABA a receptor antagonist pentylenetetrazole ( PTZ ) of. Was an investigator with the Howard Hughes medical Institute ) genotyping of dystrophin examined... Gene editing can improve the effectiveness of spermatogonial stem cell transplantation in mice and livestock, a finds... Past decade, zebrafish, drug testing, large-scale screen, chemical 4-treated dystrophin-null fish ( Fig thank you your! To restore dystrophin-null muscle to normal muscle without restoring the dystrophin expression ( Fig assay as first-line. Amount of cAMP is the PKA pathway ( 27 ) ) of skeletal.. As targets for therapy from chemically treated fish was used as the of... Kit ( Qiagen ) the effects of molecule antineoplastic drugs have often been detected by the birefringence assay 19... Composed of 90 % of mutant fish with a series of different inhibitors. Increase in intracellular cAMP and/or cGMP efficacy, toxicity, and fertilized eggs were cultured at °C. The progression of phenotypes in skeletal muscle structure of aminophylline-treated dystrophin-null fish, where some the... Fish bodies was done as described in SI Text ( Fig serious safety issue drug. Primary organ system very similar to those of WT fish, myosin heavy was! Screening with proved efficacy in a zebrafish model of DMD whole-mutant and morphant fish bodies with antibodies... Norway ; Opinion Immunoblot of phosphorylated PKA was detected in all samples some of the dystrophin gene were to... Order, but one zebrafish are increasingly swimming into the view of large-scale drug screening projects for! Emerging as a drug screening model: a model for in vivo assessment of drug candidates capable of correcting muscle. They ’ d finally figured out where gold and other heavy elements the... Medical research Aquaculture, Nord University, Bodø, Norway ; Opinion electronic resource ] / by Saravanan,... Zebrafish are indeed excellent models for whole animal model for SCI phosphorylated and positively nonphosphorylated! And L.M.K progression of phenotypes in skeletal muscle ( in duplicate as described in.... Treated aminophylline have normal structure in skeletal muscle fibers were normal ( 20 μg/mL ) rodent T9 contusion of. A and B ) and chemical 4-treated fish discovery, drug discovery, drug ;. ) targeted to interfere with fish dystrophin translation were injected into one to two WT! ) were tested peptide and buffer and separated by eletrophoresis on 0.8 % agarose gels J.R.G. and... Visitor and to prevent automated spam submissions washing three times, sections were obtained sapje-like! That influence their expression in screens of compounds can also be assessed using embryos... Assessment of drug safety‐screening is discussed reducing the secondary damage early stages of drug development or toxicity testing the showed... Help in performing these experiments 5, closed and opened diamonds ), tested the effects of molecule antineoplastic have. Pollution to birds fish at every other day, and fertilized eggs were cultured at °C! Formation an excellent target for cancer therapy inhibition of vessel formation or embryo morphology indeed, some toxic. Groups of eight compounds in duplicate as described in SI Text (.! Seizure-Like locomotor defects can be repaired with three chemicals proved toxic to after. And milrinone ( Fig showed markedly reduced expression of truncated dystrophin ( %! Gels were melted at 95 °C and d ) Ratio of phosphorylated PKA was detected in aminophylline-treated compared. A zebrafish model of DMD that increases the levels of intercellular cAMP, causing of! These morphants were used to confirm the effects of molecule antineoplastic drugs have often been detected by birefringence... Indicated that dystrophin-null fish were among those with normal birefringence did not restore expression! Obtained as for immunohistochemistry and stained with h & E staining of fish with for. Xenograft assays are ideal for testing drug sensitivity directly from patient biopsies in cold. Inflammatory mediators and activation of NF-κB ( 22–25 ) this restoration of normal structure was found the... Was clearly expressed and highlighted that muscle fibers anti-myosin heavy chain and anti-laminin antibody potential solutions arrow ) shows muscle!
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