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Zebrafish: an emerging model in preclinical research
Zebrafish are emerging as a powerful pre-clinical model for research. Some key advantages of the zebrafish model include their small size, high fecundity, rapid development and transparency during early stages. These properties allow zebrafish to be used for high-throughput drug screening, toxicity testing and studies of development. Specifically, zebrafish are useful for assessing drug effects on processes like angiogenesis, apoptosis and protein synthesis. Transgenic zebrafish lines that model human diseases also enable anti-cancer drug discovery and studies of drug resistance. Overall, the zebrafish model provides insights that can help optimize drug candidates and reduce costs prior to mammalian studies.
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Zebrafish: an emerging model in preclinical research
- 1. Zebrafish: An emerging modelin pre-clinical research Anirban Chakraborty, PhD Nitte University Centre for Science Education and Research Mangalore-575018 nucser.nitte.edu.in Pharmaquest 2021, 2-5 August,21 Tiny but mighty
- 2. Named for itsfive uniform horizontal blue stripes Belongs to the family Cyprinidae (minnows and carps) Can regenerate their skin, fins, and heart Size: Up to 4cm Life span: Usually 2-3 years, may go up to 5 years Habitat: Streams, canals, rice fields in India, Pakistan, Nepal, Bangladesh, and Myanmar Pharmaquest 2021, 2-5 August,21 Zebrafish Danio rerio
- 3. Pharmaquest 2021, 2-5August,21 http://www.ezrc.kit.edu 3Rs philosophy towards animal research Replacement: Larval zebrafish (<5 dpf) can replace animal toxicity studies Reduction: Larval zebrafish can be used as first-tier model for identifying toxic drug candidates Refinement: Embryonic transparency of larval zebrafish allows non-invasive observation of toxicities Zebrafish is a popular non mammalian alternative
- 4. Founding Father of ZebrafishDevelopmental and Genetic Research George Streisinger (1927-1984) University of Oregon, USA CHRISTIANE NÜSSLEIN-VOLHARD (1942- Pioneer in Forward Genetic Screens in zebrafish Max Planck Institute for Developmental Biology, Germany People who made zebrafish famous ! Pharmaquest 2021, 2-5 August,21
- 5. 1 2 3 4 5 6 7 Small size High fecundity Shortgeneration time Embryonic transparency Rapid ex-utero development Amenability to HTS technology Low set-up cost What makes zebrafish the preferred choice? Pharmaquest 2021, 2-5 August,21
- 6. ☼ Zygote (0- 45min) Cleavage (45 min~2h) Blastula (2.25h~4.7h) Gastrula (5h~10h) Segmentation (10h~24h) Pharyngula (24h~48h) Hatching (48h~72h) Free swimming larvae (72h ~) ⊩ ⊩ ⊩ ⊩ ⊩ ⊩ ⊩ ⊩ Early developmental stages Pharmaquest 2021, 2-5 August,21
- 7. shield (6h) 90%epiboly (9h) 3 somite (11h) 18 somite (18h) blastoderm margin * yolk plug eye primordium 4-cell (1h) 8-cell (1.25h) sphere (4h) yolk blastomere somites 〉 Pharmaquest 2021, 2-5 August,21
- 8. Day 1 Forebrain Midbrain Hindbrain Eye 4th ventricle Oticcapsule Notochord Somite yolk heart liver intestine swim bladder Day 2 Day 3 Day 5 mouth Caudal fin Pharmaquest 2021, 2-5 August,21
- 9. Zebrafish Development For avertebrate, extremely rapid and occurs ex-utero Transparent, each stage can be easily observed Pharmaquest 2021, 2-5 August,21
- 10. Pharmacology Toxicology Functional Genomics Disease Modeling Zebrafish in biomedical research Zebrafishmodel system: A powerful tool Egg Embryo Adult Pharmaquest 2021, 2-5 August,21
- 11.
- 12. Usefulness of zebrafishmodel Drug Discovery Assessment of Drug-induced Toxicity Pharmacology Pharmaquest 2021, 2-5 August,21
- 13. December 17, 2019 Pharmaquest2021, 2-5 August,21
- 14. Drug discovery: Theinitial steps Chemical Screen/ Small molecule screen Bioactivity Toxicity Off target effects Identification of Lead Compounds Onset of drug development Pharmaquest 2021, 2-5 August,21
- 15. Strategies for Drugdiscovery Bowman and Zon, 2010 Pharmaquest 2021, 2-5 August,21
- 16. Zebrafish in drug discovery EMBOreports, 2009 Pharmaquest 2021, 2-5 August,21
- 17. High Throughput Screening(HTS) •A drug discovery process commonly used in the pharmaceutical industry •An automated technique to quickly assay the biological or biochemical activity of a large number of drug-like compounds •Typically, HTS assays are performed on “automation-friendly” micro- titre plates with a 96, 384, or 1536 well format Pipetmax A 96 well microtiter plate Pharmaquest 2021, 2-5 August,21
- 18. Zebrafish Whole-animal based phenotypicassay Scalability of in vitro systems 4 day old zebrafish in 96-well plate Can survive without food for 7 days Embryos are few millimetres in length Tolerant to DMSO and readily absorbs compounds from water Adults can be treated with compounds via intraperitoneal or intracardiac injections Pharmaquest 2021, 2-5 August,21
- 19. The optical transparencyof the embryos allows in vivo visualisation of expression patterns of genes and proteins Tracing of xenografts Whole organism-based “readouts” Advantages of zebrafish Pharmaquest 2021, 2-5 August,21
- 20. mRNA in situhybridization Antibody staining Visualization of fluorescent reporter DNA/RNA or tagged xenografts Whole-mount fixed embryos Live whole embryos Pharmaquest 2021, 2-5 August,21
- 21. WISH (whole-mount insitu hybridization) Hindbrain-specific transcription factor hindbrain midbrain forebrain Brain-specific transcription factor bop1 midbrain eye posterior somites Ubiquitous nucleolar protein gata1 intermediate cell mass Erythroid-specific transcription factor posterior blood island Pharmaquest 2021, 2-5 August,21
- 22. Antibody staining p-Histone H3staining mitotic/proliferating cell marker Pharmaquest 2021, 2-5 August,21
- 23. Terminal deoxynucleotidyl transferase(TdT) TUNEL assay TMR-labeled nucleotides Enzymatic detection of labeled nucleotides Single strand breaks (nicks): free 3’-OH ends apoptotic cell marker O-dianisidine (ODA) staining Hemoglobin catalysis + H2O2 Conversion of ODA chromogen red blood cell marker in vivo labeling of catalyzing enzymes Pharmaquest 2021, 2-5 August,21
- 24. Genome manipulation tools 43493fish lines http://zebrafish.org/zirc Gut et al, Physiol Rev 2017 Pharmaquest 2021, 2-5 August,21
- 25. Zebrafish in Anti-cancer DrugDiscovery Pharmaquest 2021, 2-5 August,21
- 26. Transgenic/mutant zebrafish modelsof organ-specific human cancers Over expression of oncogenes in specific tissues Develop cancer in target tissue Whole-animal Drug screening Pharmaquest 2021, 2-5 August,21
- 27. For instance, Whole animaldrug screen for liver cancer drugs Liver specific Tg line (lfabp: kRAS-mcherry: HCC) Pharmaquest 2021, 2-5 August,21
- 28. WA-HTS for livercancer drugs X Human disease models (Liver cancer) Array embryos/larva to multi-well plates Functional ingredients from foods Chemicals Bioactive compounds from the environment Phenotypic assessment Biochemical assays (ISH/IHC for markers) rescue IDENTIFY POTENTIAL CANDIDATES Pharmaquest 2021, 2-5 August,21
- 29. Similarly, Screen for inhibitorsof nascent protein synthesis Untreated Treated with drugs Reduced Alexa488 signal in the treated embryos indicating a reduction in nascent protein synthesis Pharmaquest 2021, 2-5 August,21
- 30. Screen for inducersof apoptosis Untreated Treated with drugs Increased TM Red signal in the treated embryos indicating extensive apoptosis Pharmaquest 2021, 2-5 August,21
- 31. Or, Screen for inhibitorsof angiogenesis Untreated Treated No difference in fli-1 expression between the treated and untreated embryos, indicating no effect of the drug on angiogenesis Pharmaquest 2021, 2-5 August,21
- 32. Xenotransplantation assays foranti-metastatic drugs White et al Nature Reviews, 2013 Pharmaquest 2021, 2-5 August,21
- 33. New use ofzebrafish xenograft models Patient-specific chemosensitivity profile based on Tumor Cell Behaviour (Proliferation, Angiogenesis, Migration and Metastases) Xiao et al, Trends in Cancer, 2020 Personalized Zebrafish Xenografting Pharmaquest 2021, 2-5 August,21
- 34. Drug Resistance Modelingin Zebrafish Identification of potential drug-targets for drug-resistant cancers Pharmaquest 2021, 2-5 August,21
- 35. A small moleculescreen for HSC enhancers Cell, Communication and Signalling, 2010 dmPGE2 Stimulates HSC development in zebrafish adults and embryos Also in Mouse Treatment of umbilical cord blood with dmPGE2 prior to transplant A success story from zebrafish Pharmaquest 2021, 2-5 August,21
- 36. Untreated/Unexposed Developmental delayDeath Treated/Exposed Zebrafish in Assessment of drug-induced toxicity Nature Protocols, 2009 Pharmaquest 2021, 2-5 August,21
- 37. Cassar et alChem Res Toxicol, 2020 An upward trend Pharmaquest 2021, 2-5 August,21
- 38. The story of“failed drugs” Drugs that are promising but terminated due to low efficacy, adverse side effects, and excessive toxicity Thalidomide: • Extremely valuable, first used in Europe • During 1958-1962, used as a sedative for pregnant women (first trimester) • But causes severe developmental defects, phocomelia most characteristic •> 10,000 babies affected, approx. 40% died during infancy Phocomelia (malformed due to absence of some or all of the long bones,with retention of digits giving a ‘flipper’ or ‘seal-like’ appearance) Pharmaquest 2021, 2-5 August,21
- 39. Proof of Principle Cell,Communication and Signalling, 2010 Thalidomide treatment in zebrafish Pharmaquest 2021, 2-5 August,21
- 40. in vivo model Drug Absorption Distribution Metabolism ExcretionToxicity assessment Advantages of zebrafish Prioritize drug candidates by eliminating potentially unsafe compounds during early stages of drug development. Can reduce unnecessary costs for mammalian studies. Can serve as an an intermediate step between cell-based evaluation and conventional animal testing. Pharmaquest 2021, 2-5 August,21
- 41. zebrafish larvae An adultzebrafish Cassar et al Chem Res Toxicol, 2020 White et al Nature Reviews, 2013 Developmental toxicity Neurotoxicity and behavioral analyses Ocular toxicity Intestine, pancreas and hepatobilary toxicity Nephrotoxicity Endocrine toxicity Hematologic toxicity Cardiovascular toxicity Ototoxicity Pineal/circadian rhythm Pharmaquest 2021, 2-5 August,21
- 42. Assays for toxicity 1.Developmental toxicity 2. Cardiotoxicity 3. Hepatotoxicity 4. Neurotoxicity Pharmaquest 2021, 2-5 August,21
- 43. Toxicological end points Lethal: •Noheartbeat Sublethal/Teratogenic: •Malformation of heart •Defective cardiac functions (heart rate, rhythmicity, circulation) Cardiotoxicity Visualisation of heart using fluorescent markers Pharmaquest 2021, 2-5 August,21
- 44. Day 3 (72hpf): Hatching efficiency analysis Nanomaterial/Drug Untreated In zebrafish: maximum hatching by day 2, complete by day 3 Either premature hatching or inhibition of hatching and embryonic death within chorion Pharmaquest 2021, 2-5 August,21
- 45. Visual assessment ofliver necrosis Nanoparticle/Drug/Chemical Untreated Pharmaquest 2021, 2-5 August,21
- 46. Neurotoxicity and behaviouralresponse Pharmaquest 2021, 2-5 August,21
- 47. MSc thesis, Univ of Oslo, 2015 Zebrabox: Automatedbehaviour response system Pharmaquest 2021, 2-5 August,21
- 48. Evaluation of Genotoxicity, OxidativeStress and Cytotoxicity Pharmaquest 2021, 2-5 August,21
- 49. Oxidative Stress (ROS)and DNA damage- commonly associated with nanoparticles Nrf2 antioxidant pathway conserved in zebrafish Fold change in sod1 Pharmaquest 2021, 2-5 August,21
- 50. in vivo detectionof ROS in zebrafish Expose to nanoparticles/drugs at different concentrations Array embryos/larva to multi-well plates H2DCFDA Process of oxidation converts a Non-fluorescent H2DCFDA Fluorescent H2DCFDA Incubation for 2 hours in dark Wash and observe under fluorescence microscope Pharmaquest 2021, 2-5 August,21
- 51. Pharmaquest 2021, 2-5August,21
- 52. Zebrafish: a powerfultool •Becoming the model of choice for biologists, toxicologists, pharmacologists •Great potential as an alternative vertebrate model •Many commercial companies have brought in zebrafish into their portfolio •Despite limitations, zebrafish can effectively complement other mammalian and non-mammalian model systems Pharmaquest 2021, 2-5 August,21
- 53. THANK YOU Zebrafish Group Divisionof Molecular Genetics and Cancer, NITTE-Deemed to be University, India • Dr. Gunimala Chakraborty- Faculty • Mrs. Rajeshwari Vittal- Faculty • Ms Dechamma P N- PhD student • Mr Nishith B- PhD student • Ms Krithika K- PhD student • Ms Sonam K- PhD student • Ms Nidhi S- PhD student • Ms Mrunali Devadiga- JRF • Ms Athira Viswambharan- JRF • Ms Anjali - MSc student • Mr Shivkiran- Intern • Mr Rasik- BSc student • Mr Mithun- zf technician