Credit: The University of Manchester
As some of the most used experimental animals after mice, studies using zebrafish have been at the forefront of some of the most important biomedical research in recent decades. While it may seem surprising, these small, tropical, freshwater fish can provide us with incredible insights into diseases and conditions that also affect humans – from cancer to diabetes to brain injury – and give researchers the chance to develop and test new techniques that can ultimately benefit human health.
One reason zebrafish are so widely used is because painstaking research has revealed the complete set of zebrafish genes (whole genome), and has meant that we can clearly see just how many of the same genes that zebrafish and people share.
In 2013 the zebrafish genome project, conducted by the Wellcome Sanger Institute, UK, found that 70% of all genes are shared, while 84% of zebrafish genes have a link to human disease.
Since the late 1990s, when their use as a research animal became more common, zebrafish have time and again demonstrated their value, providing crucial insights into how diseases work and how to treat them. Now, they are proving to be ideal models for use with new technologies that have the potential to transform aspects of human healthcare.
In this feature, we will outline the use of zebrafish in biomedical research worldwide, including what makes them useful as a research animal, the research areas in which they play an important role, as well as how their use is changing.
This [zebrafish] genome will allow researchers to understand how our genes work and how genetic variants can cause disease in ways that cannot be easily studied in humans or other organisms.
Dr Derek Stample, Wellcome Sanger
Zebrafish research facility (Credit: The University of Manchester)
Why do we use zebrafish in biomedical research?
Researchers may choose to use zebrafish over mammals, and other species of fish, for a range of different reasons.
In research, it is always preferable to use animals that are considered lower on the animal hierarchy in terms of their complexity, where appropriate – for example, zebrafish are generally preferred over mice, and mice preferred over monkeys.
The transparency of zebrafish is extremely useful for studies into the development of embryos, or the understanding of the toxic effect of substances, to clearly see how the body is structured, or affected. For example, EARA member the Champalimaud Foundation, in Portugal, uses zebrafish larvae (young fish in the early stage of development in its research into the formation of circuits of neurons.
See also this EARA Q&A video on the use of zebrafish embryos (at the earliest stage of development in the egg) for chemical safety assessment of the anti-epileptic drug, carbamazepine.
The similarity of zebrafish genes with the human genome not only make zebrafish appropriate for studying human genetic conditions, but also for genetic modification. For example, mutations can be introduced into the zebrafish genes of interest to study the effect this will have on specific diseases, including rare ones.
Researchers at University College London, UK, and Semmelweis University, Hungary, identified the gene linked to kidney disease in zebrafish, as well as recreated the condition in the animals.
Genetic modification has also been made easier and more efficient with the rise of powerful gene editing tools like CRISPR-Cas9, including work at Eötvös Loránd University, Hungary, which used this technology to develop a zebrafish model of a rare genetic disease that can cause blindness.
Zebrafish live in water and it is easy to modify their genes simply by adding different substances to their tank, as these then get absorbed by the fish. This also makes it simpler to look at the effect of drugs and chemicals on the body and observe their effects.
It is relatively straightforward to directly transplant cells into zebrafish embryos. And as zebrafish lay their eggs outside the body, it is much easier to access, manipulate and observe the embryos. Some of the research areas that use zebrafish embryos include embryonic development and stem cell biology.
The ability of zebrafish to regenerate their limbs and organs, including the heart, can give new insights into how injury and healing work, for instance, to develop regenerative medicines for humans (see next section).
Zebrafish possess a backbone like humans which makes them suitable for studies of the body’s organisation and function, and as a replacement for a mammal such as a mouse. It means that zebrafish are a useful model to study, for example, the development of the nervous system.
We selected the zebrafish because it carries a version of the gene that is very similar to that of humans, making it a great model system to study.
Dr Jennie Chandler, UCL
Zebrafish larvae (Credit: The University of Manchester)
Zebrafish (Danio rerio) are freshwater fish that grow up to two inches in size, with characteristic blue horizontal stripes across their body. They are found in a variety of habitats in large social groups and originate from India.
An embryo is the one of the earliest stages of a zebrafish, when it is still developing inside the egg (see image right). For laboratory strains of zebrafish, embryos hatch into larvae three to four days after the egg has been fertilized. The swimming, feeding larvae will then take around six weeks to develop fully into adults.
Because zebrafish are small in size, have a short lifespan, and reproduce quickly and in large numbers (females can lay hundreds of eggs in one week), they have many of the qualities that make them appropriate for use in a research setting.
What is a zebrafish?
Zebrafish embryos. (Credit:) Max Planck Institute for Developmental Biology)
Compared to mice, it can also be easier to keep zebrafish in the lab thanks to their flexibility in diet, water quality and habitat.
Zebrafish have transparent larvae and embryos, unlike many other animals typically used in research, such as mice. This means that their organs and body systems can be easily imaged and visualised (without needing high-resolution microscopes). The transparency of zebrafish also avoids the need for intrusive procedures that may injure the fish.
These elements have enabled researchers to make breakthroughs using zebrafish in areas such as genetics and cell development – read this Your Genome article on milestones in zebrafish research.
A global consortium, involving EARA member the Max Planck Institute (MPI), in Germany, also recently annotated the zebrafish genome to produce the largest genetic atlas to date.
Where has research with zebrafish made a difference?
From new knowledge into diseases and health to testing drugs and treatment strategies, scientists are beginning to understand more about how zebrafish can contribute to research.
Here are some of the main areas, both established and more recent, that have made use of zebrafish.
Embryology and development
Studies into how embryos form, grow and develop – and what happens when it goes wrong – have benefited hugely from the use of zebrafish, helping researchers to understand the key genes involved in these processes and the factors that lead to developmental abnormalities.
For instance, in a study at Augusta University, Georgia, USA (then the Georgia Health Sciences University), scientists were able to confirm that the PHF21A gene was involved in head and brain abnormalities in the rare disorder Potocki-Shaffer syndrome, by deactivating the gene in zebrafish.
Now that we know the causative gene, we can sequence the gene in more patients and see if they have a mutation.
Dr Lawrence Layman, Augusta
When it comes to conditions related to the brain, zebrafish have been used to not only improve our understanding of neurological conditions, such as motor neurone disease (MND) and multiple sclerosis (see this University of Edinburgh, UK, study), but also behaviour (see next section). This is because the zebrafish brain and nervous system can be studied at the level of nerve cells, in both healthy and diseased tissue, to reveal new information about how these conditions work and, in turn, can be treated.
Forms of MND, such as amyotrophic lateral sclerosis (ALS), currently lack a cure, and symptoms tend to get progressively worse as the motor neurons in the brain and spinal cord die. A study, using zebrafish, at the Institut National de la Recherche Scientifique, Canada, revealed the differences in the neural system between healthy and diseased zebrafish with ALS-like symptoms, to help understand how ALS develops in a non-harmful way.
Zebrafish can also accurately mimic strokes, for example in the research by EARA member the University of Manchester, UK, which developed an improved way to study haemorrhagic stroke (brain bleeding that kills brain cells) that closely resembles the condition in humans.
Because zebrafish show similarities with humans in how they behave, such as in terms of social behaviour, they can be very useful in the understanding of learning, cognition and brain function, as well as conditions related to these factors, such as stress and anxiety.
Zebrafish also have several of the same brain chemicals to humans - those that control behaviour - including oxytocin, which is involved in emotions like love and fear.
In a study at EARA member the Max Planck Institute for Biological Intelligence, for instance, researchers were able to selectively breed zebrafish to uncover distinct personality traits and brain activity differences associated with human psychiatric conditions.
Front of zebrafish (Credit: University of Bristol)
Meanwhile, scientists at Harvard University, Massachusetts, USA, gained surprising new insights into behaviour during feeding by studying zebrafish larvae.
Larvae have also revealed how social experiences can impact behaviour at an early stage, in research by EARA member the Champalimaud Foundation, Portugal, to potentially shape behaviour into adulthood.
The pace at which personality traits can be shifted and fixed in evolution [in zebrafish] is remarkable.
Dr Herwig Baier, MPI for Biological Intelligence
Zebrafish can develop similar cancers to humans, such as skin cancer, with tumours that can resemble those found in cancer patients. One of the ways to do this is through a xenograft – where tissue from one species (in this case, humans) is grafted or implanted into a zebrafish (an avatar) even though the zebrafish will not have this cancer naturally. These cancer avatars can then provide evidence about how well a particular drug, or chemotherapy, may work in the clinic, without the unknown risk of testing it in people.
The clear bodies of zebrafish larvae can allow for more precise imaging techniques to be developed, or for live tracking inside the body, such as to monitor the development of tumours, or the passage of toxins and their resulting effect.
Stain of a zebrafish larval brain. Credit: Ruth Diez del Corral, Champalimaud Foundation
Some of the main advantages to using zebrafish in general – namely, a fully sequenced genome that shares many genes with humans, and transparent embryos – also means that scientists can search for genes within the zebrafish genome that are linked to human cancer, and precisely visualise cancer growth at the level of single cells. This also applies to drug discovery by testing small molecules for their response to treatment, or toxicity, such as the study at the zebrafish core facility of the Karolinska Institute, in Sweden.
Recently, researchers at the Children’s Hospital Los Angeles, USA, were also able to reliably study the aggressive childhood bone cancer Ewing sarcoma by using zebrafish, to shed light on how the tumours grow and interact with other cells and molecules in the body.
It’s revolutionized our approach as a laboratory. We can now look in an unbiased way to find unexpected new insights into what drives tumour growth.
Dr James Amatruda, Children’s Hospital LA
In the EU in 2020 (plus Norway), zebrafish were the fourth most common animal used in scientific research, making up 277,328 of the total number (3.5%). They were the second most genetically altered animal, behind mice, with 23% of all zebrafish being modified for the purposes of research.
The use of zebrafish in the EU has been steadily rising over the years – for example, there was a 12% increase in the EU from 2018 to 2019. One reason for this increase is that these animals are beginning to replace other animals such as rodents, under the principles of the 3Rs (replace, reduce, refine) in research. This is particularly true when using zebrafish embryos instead of adult animals – read our feature on the 3Rs in animal research.
There are plans to use zebrafish embryos as a replacement for mice when researching some of the genetic mutations involved in depression, in an upcoming study led by King’s College London, UK.
As technologies, such as gene editing and artificial intelligence (AI), develop and become more widely available, the research community may increasingly expect to utilise zebrafish in their fields of work, including precision medicines that are tailored to a specific patient for treating cancer and neurological diseases.
How widely are zebrafish used in Europe?
Metabolic diseases and other conditions
Conditions affecting the metabolism (the chemical reactions that provide energy in organisms) such as obesity and diabetes, are among the most prevalent – and growing – global health conditions today. As zebrafish are similar to humans in several key aspects related to metabolism, such as the biology of fat cells and tissue, they can be used to study these types of disease to understand the effects of genetic and environmental factors, or to identify new targets for treatments.
At the Karolinksa Institute, Sweden, researchers found a molecule that can stimulate the formation of cells that produce insulin – a crucial hormone needed to manage type 1 diabetes – to lower blood sugar levels in zebrafish. While another team, at the University of Sheffield, UK, developed a method to observe how the brain regulates blood supply in zebrafish, which not only led to the discovery of a drug that can reverse the effect of high glucose in diabetes on the brain, but also new insights into Alzheimer’s disease.
The results are really promising and pave the way for further diabetes research in this area using zebrafish.
Dr Clare Howarth, Sheffield
Zebrafish have also provided important insights into diseases caused by infections, such as tuberculosis – a group at Leiden University, Netherlands, identified how the bacteria can be cleared most efficiently from the lungs of zebrafish by looking at the movement of specific immune cells.
Zebrafish can even help to understand how spaceflight affects humans. In research by Queen’s University Belfast, UK, zebrafish in a hibernation state were found to experience fewer effects from radiation, bolstering our knowledge of how humans may safely travel to Mars.
Zebrafish can regenerate many of their organs and body parts, including the heart, nervous system, eyes and fins and they could hold the key to understanding how regeneration could also be achieved for people with permanent injuries, such as from accidents, or lasting damage caused by conditions like cardiovascular disease and diabetes.
A team at the Weizmann Institute of Science, Israel, has looked at the healing powers of zebrafish to understand how they are able to regenerate their heart. And a group led by Imperial College London, UK, has uncovered a vital mechanism in zebrafish embryos for the development of heart valves – see the video below.
Heart development in a healthy zebrafish embryo (L) and in an embryo with a mutation (R). Credit: MPI for Heart and Lung Research
Meanwhile, a study at Ludwig Maximillian University (LMU), Germany, used zebrafish to understand how to prevent the formation of scars in the brain that result from brain injury.
The idea was to tease out the differences between zebrafish and mammals so as to understand which signalling pathways in the human brain inhibit regeneration – and how we might be able to intervene.
Professor Jovica Ninkovic, LMU
Nanomedicine and new technologies
This is a step.. toward cures for some of the devastating brain disorders faced by humans.
Professor Joseph Fetcho, Cornell
Zebrafish have also proven to be an ideal organism to use to improve and refine new scientific techniques - an example is imaging the body using microscopy and other methods. Although transparent when younger, zebrafish become more opaque as adults, but researchers have worked to overcome this, for example at Cornell University, New York, USA, which developed techniques to allow the networks of whole adult zebrafish brains to be visualised to better study brain disorders.
Meanwhile, a study at the Swiss Federal Institute of Technology in Lausanne (EPFL), Switzerland, tested a microsurgeon robot on zebrafish embryos to demonstrate that the tool could successfully target specific areas of tissue to look at how a body grows.
Another emerging scientific field is the use of medicines at the smallest cellular scale possible (nanomedicine), which will allow substances to pass through certain barriers in the body that larger drugs cannot. In this way, nanoparticles can also be used to deliver drugs into the body.
As zebrafish have similar organs and blood systems to humans, they are a good option to assess how nanomedicines work when they are inside an organism, as well as possible side effects.
It is also important to know how these tiny particles may behave once they are in the body – a study at Aarhus University, Denmark, for instance, has traced the journey of nanoparticles through zebrafish embryos to assess how well they reached their target of solid tumours.
Credit: University of Bristol
How are zebrafish looked after in research?
As part of the 3Rs principle, researchers are continuously working on ways to use fewer zebrafish. Some of the key ways this can be achieved is by improving housing conditions, for example by enriching tanks with plants to encourage natural exploring behaviours, and keeping zebrafish in social groups, both of which can also minimise distress and anxiety.
When it comes to experimental procedures, any pain that zebrafish may experience can be kept to a minimum by using different types of anaesthesia, depending on whether they are adult zebrafish or embryos. These measures also mean that fewer zebrafish can be used in the long run, because the standard and repeatability of experiments will be improved.