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More than just a fish: the power of zebrafish in research

Since its debut in the early 80s, the use of Danio rerio (zebrafish) in biology has grown so fast that it is nowadays the second most used animal model in research. Indeed, although zebrafish might appear evolutionarily very far from us, approximately 70% of human genes have an ortholog in zebrafish. In fact, orthologs are genes that derive from a common ancestor and that codify for the same protein. Here we focus on some advantages that Danio rerio brings to the study of vertebrate development and disease, as well as its application in different fields of science.




The importance of animal models in science


Before entering into the merit of this article it is anyway important to explain why nowadays the use of animal models in science is still necessary. In vitro models, like 2D cells culture or 3D organoids, have improved significantly in the last years but, unfortunately, they present some limitations that make the use of animals in experimentation fundamental. First of all, we should recall the remarkable anatomical and physiological similarities between humans and animals, particularly mammals; this allows, for example, a better comparison in a pathological context. Secondly, animals are very complex organisms in which organs achieve distinct physiological functions thanks to a complex network of hormones, circulating factors and cells and cross-talk between cells in all the compartments. This means that the use of these models, like the zebrafish one, is of primary importance to understand how biological processes happen in a complex and full organism. 

Of course, animal experimentation is strictly supervised and each experimental design has to be approved to be sure that it follows the rule of the three Rs: reduce, refine and replace. The researcher should initially try to replace, wherever they can, the animal model with an alternative one; the second step is to try to reduce as much as possible the number of individuals used in a certain experimental protocol; finally, the last R refers to the operation of refining, or improving, the experimental conditions to which the animals are subjected.


General characteristics of the model


Figure 1. Male and female zebrafish


These fresh water fish are native to India and South Asia, and owe their name to the blue stripes which horizontally cross their bodies. This species is oviparous; one female is able to lay between 200 and 300 eggs after each crossing. This is appealing when considering the necessity of including many animals in your experimental set-up. The first stages of development are fast, with the embryo hatching from the egg at two days post fertilisation (dpf), transitioning into the larval stage at five dpf, and finally juvenile at 30 dpf. Sexual maturity is reached at three months while the zebrafish lifespan in captivity is about two to five years. Individuals do have visible differences between male and female; males generally have a thinner, goldish coloured body while females are larger (especially if carrying eggs) and have a silverish colour. Among the characteristics that make this model appealing, we note the generation of hundreds of eggs in a single clutch and the transparency of the developing embryos, which allows for live imaging at the organismal level. Finally this model allows for the possibility of creating animals carrying in their genome external DNA (transgenic animals), which can be used to study the roles of shared genes.



A few examples of zebrafish use for biomedical research


Zebrafish models of brain disorders and addiction


The zebrafish has recently been used to investigate debilitating neurobehavioral disorders including motivational and social impairments, such as depression and autism spectrum disorder (ASD). Their use in these fields is supported by several behaviours which can be used as evidence in neurobiology, enabling the measurement and study of social interactions or social preference. For example, a robust behaviour exhibited by fish is the tendency to form tight groups (shoaling). Abnormality in this phenomenon can be considered as a sign of atypical social behaviour. In addition, both larval and adult zebrafish show high sensitivity to different addictive drugs commonly abused in human society, such as tolerance, clear preference for these agents and withdrawal symptoms. The effects of alcohol in zebrafish have been studied for more than a dozen years, and have revealed numerous behavioural changes in fish that resemble those seen in rodents and humans.



Regeneration ability


Another strength of the zebrafish model lies in its regenerative capacity. Understanding the mechanisms underlying organ regeneration using this model system might open the door for future applications in the field of tissue repair. Tissue damage can be resolved by healing and regeneration, however different species respond in different ways. The capacity for tissue regeneration varies widely across vertebrates; Danio rerio is well-known for being able to regenerate different tissues and organs including the spinal cord, brain, retina, hair cells, muscles, fins, skin, internal organs and more. Genetic tools are also available in zebrafish, and can aid in clarifying the role of the genes involved in regeneration.


Disorders of the musculoskeletal system


The identification and validation of key genes and potential targets in pathologies of the bone, tendon, muscle and cartilage have expanded the usefulness and relevance of the zebrafish model in orthopaedic research. The fully developed skeleton is composed of several functional groups; this includes the cranial musculoskeletal system, which forms rapidly and can function by 5 dpf. Different pathologies can be mimicked and studied using this animal model: hereditary collagen diseases, scoliosis, osteoarthritis, and more. Zebrafish typically grow throughout life and there is evidence that skeletal function declines with age. For instance, the mechanical properties of tendons diminish with age and alterations of vertebral bone and disc are observed. Considering that certain debilitating conditions arise in the skeleton with ageing, such as osteopenia and osteoporosis, the ability of the zebrafish to model components of these processes might be important. With more analyses of late adult stages, we may gain more insight from the zebrafish into how the skeletal system ages and its consequences.


Limits of the model


Despite the strengths described above, the zebrafish model has some limitations. For example, a recognized limit in zebrafish genetics is the paucity of well-characterised inbred strains. We use this definition to refer to individuals of a particular species which are nearly identical to each other in genotype due to long inbreeding (mating between individuals that are genetically related). Continual inbreeding produces mice that are genetically uniform and in addition to this, these strains are well characterised with regard to their physiology, how they respond to experimental perturbations, and what to expect as they age. Importantly, the use of inbred strains can also significantly reduce the number of animals needed for each experiment. Unlike mouse models, where almost 100 well characterised inbred strains and hundreds of mutants are available to researchers, generation of well characterised zebrafish mutants is still in its early stages. While zebrafish are highly social shoaling animals, it is unclear if deficits in shoaling behaviours can be compared to complex human ASD-like phenotypes. Further, different behavioural tests which are commonly used in mice are still being adapted to fish. In the context of zebrafish for orthopaedic research this field is still in its relative infancy and there are a number of open questions regarding developmental stages, bones, and phenotypic traits that are still unresolved. There are also some morphophysiological differences that can make one-to-one modelling of human skeletal phenotypes in zebrafish challenging. While origins of mammalian bones and their connections to fish bones can sometimes be identified through evolutionary analyses, such connections cannot always be made.

Despite its limitations, the zebrafish model is undeniably useful and important in some key biological research. The zebrafish represents an animal model able to recapitulate many human diseases, with its own distinct advantages in comparison to other common animal models.


References


  1. Busse, Björn, et al. (2020) Zebrafish: an emerging model for orthopaedic research. Journal of Orthopaedic Research. https://doi.org/10.1002/jor.24539

  2. Kalueff, A. V., et al. (2014). Zebrafish as an emerging model for studying complex brain disorders. Trends in pharmacological sciences. https://doi.org/10.1016/j.tips.2013.12.002

  3. Marques, I. J., et al. (2019) Model systems for regeneration: zebrafish. Development. https://doi.org/10.1242/dev.167692

  4. Choi, T. Y., et al. (2021). Zebrafish as an animal model for biomedical research. Experimental & Molecular Medicine. https://doi.org/10.1038/s12276-021-00571-5

  5. Schmidt, S. J., et al. (2011). Social cognition as a mediator variable between neurocognition and functional outcome in schizophrenia: empirical review and new results by structural equation modelling. Schizophrenia bulletin. https://doi.org/10.1093/schbul/sbr079

  6. Howe, K., et al. (2013). The zebrafish reference genome sequence and its relationship to the human genome. Nature. https://doi.org/10.1038/nature12111


This article was specialist edited by Dr. Laure Bally-Cuif and copy edited by Dr.Carys Croft


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