The Zebrafish (Danio rerio) is becoming a highly popular animal model in research. There are several reasons for this choice including not only its numerous technical advantages but also a growing pressure towards the reduction of animals for research following the 3R policy (Replacement, Reduction, and Refinement). One of the reasons why this animal model is becoming very popular is because assays using Zebrafish under 5 days post-fertilization (dpf) are not considered in vivo assays according to European Directive 2010/63/EU. Furthermore, many advantages justify the use of Zebrafish research including that it is a cost-effective model, it is ready to use within 24 hours, gestation takes place out of the mother, it develops fast, it has high genetic homology with humans (sharing up to 84% of disease-associated genes), it is amenable for high throughput, and larvae are transparent being able to see any effects in their organs.
Zebrafish assays generally fall under two different categories. These fish may be used to perform efficacy assays for disease model generation and target validation purposes, and they are also often used to expose multiple toxicities of new developing drugs. Modern gene-editing technologies such as CRISPR, offer the possibility to generate transgenic Zebrafish lines that target specific pathways, diseases, and organs. These advanced molecular tools contribute to establishing solid and affordable assays to better predict the human efficacy and toxicity of novel chemicals.
General Toxicity Assays
The Fish Embryo Acute Toxicity Test follows the OECD 236 Guideline and is a rapid and inexpensive test that includes endpoints such as mortality, somites formation, heartbeat, or tail detachment.
A more comprehensive toxicity test is a teratotoxicity assay where more than 10 morphological endpoints are checked at different time points.
Furthermore, there are also ecotoxicity assays such as the Fish Embryo Toxicity (FET) test to make a risk assessment and determine the potential damage of chemicals to the environment.
Specific Toxicity Assays
The heart is along with the brain the most important organ and this is why cardiotoxicity is a crucial checkpoint. There are transgenic lines of Zebrafish that show a fluorescent heart as a result of the Green Fluorescent Protein (GFP) engineered to specifically glow this important organ. This feature, in conjunction with the transparency of larvae at their early stage, makes Zebrafish a very attractive model for predicting the cardiac toxicity of novel compounds.
The brain is the other main organ that is critical during the development of a vertebrate. Alterations in the mobility pattern of larvae induced by known drugs have been observed. This fact strongly supports the use of Zebrafish as a predictive model of neuroactivity and neurodegeneration in humans.
The liver is an organ that processes our blood and metabolizes drugs into forms that are nontoxic and easier to use by our body. Hepatotoxicity is therefore a key factor to bear in mind during the development of a new drug. Common experimental assays that study liver diseases and hepatotoxicity include the use of both in vitro and in vivo models such as hepatocytes and rats respectively. However, the increased pressure in reducing the number of animals in research, yet using more complex systems than cells has made the Zebrafish model a valuable alternative animal model.
A wide variety of compounds including drugs, fertilizers, and natural pollutants are known to have a major impact on nephrogenesis and renal functionality. There is an unmet need for nephrotoxicity assessment and the Zebrafish model offers the opportunity to elucidate also these potential human health hazards.
Other toxicity assays that complement the armamentarium of this useful animal model include assays as important as ototoxicity, thyroid disruption, or immunotoxicity. All of these assays can be carried out in a well-formed complex vertebrate model while respecting the ethical rule of not being considered an in vivo model within the five first days of life.
A successful clinical candidate needs not only to be safe but also to prove efficacious. Once a threshold dose of a particular drug has been proven to be safe, a disease model is needed to see the therapeutic effect of the candidate compound.
Target identification and validation are gaining relevance in the early phases of Drug Discovery. This process allows characterizing the role of a protein or pathway of interest and provides selection arguments to define the required properties of the compounds to be screened.
Zebrafish has been proposed as a good model to find genes involved in specific processes and/or check the activity of the desired gene in a specific process. Different strategies and tools can be used to unravel the function of a specific gene. Among them, the transient inhibition with Morpholinos (MO) is a common one. Furthermore, the Zebrafish animal model allows combining different MOs at the same time to unravel possible synergistic effects between different genes. Another state-of-the-art technology for this purpose is the use of F0 knockout Crispants allowing the direct generation of maternal-effect phenotypes.
All these tools are key to establishing efficacy assays that mimic human diseases in the Zebrafish animal model particularly in the fields of oncology, metabolism, cardiovascular, muscular diseases, rare diseases, and the central nervous system (CNS). Some of these CNS-related models and rare diseases may include Parkinson's, Epilepsy, Amyotrophic Lateral Sclerosis, Duchenne Muscular Dystrophy, Tauopathy, Huntington Diseases, or Dravet Syndrome. But there are many other therapeutic areas where the Zebrafish model might also be of interest such as the research on infectious diseases and even efficacy assays for cosmetics.
Overall, in this article, we have shown the great potential for this small fish in adding solid research tools toward the development of potentially life-saving drugs.