The small tropical fish, Danio rerio, a familiar sight in home fish tanks, has in the space of a few years gained star status in the international research community. 

BY KURT BUCHMANN ET AL

Kurt Buchmann, professor of aquatic pathobiology, Faculty of Health and Medical Sciences, University of Copenhagen. His field of research covers fish health, fish biology, vaccinology and immunology. Per W. Kania, associate professor of aquatic pathobiology. Conducts research in molecular biology tools for fish. Simon Haarder, PhD student. His research has particularly concerned intestinal functions in healthy fish. Louise von Gersdorff Jørgensen, assistant professor of pathobiology. Designs fish models for applications in fish immunology and vaccine research.

Project: ‘The Zebrafish Research Model’

Grantee: Department of Veterinary Disease Biology, University of Copenhagen

Amount: DKK 2,424,000 from VILLUM FONDEN

The project links a number of basic physiological functions in a single zebrafish model. The model will at one and the same time be capable of demonstrating an entire individual's reaction to an environmental factor. The focus is thus not merely on a single topic, but takes into account the entire functioning of the fish. This comprises the fish's immediate behavioural reaction in the spatial dimension; its internal organ functions (heart, brain, liver, spleen, glands), intestinal function, reproductive organs and immune system. The project attaches importance to presenting and viewing the fish holistically rather than as fractioned sections.

From home fish tank to research

The zebrafish, bearing the scientifc name Danio rerio, has been a popular pet fish in home fish tanks for decades (Figure 2). This little striped fish, which originated from rivers in northern and central India, has delighted young and old tropical fish enthusiasts all over the world. 

However, in the past, this seemingly ordinary fish was not the object of any notable scientifc interest. In recent years, though, researchers have discovered the value of the zebrafsh as an experimental animal. This fish can easily be kept in limited space in the laboratory as it is usually no more than 3 cm in length. It breeds rapidly in captivity, producing many juveniles (100-300 eggs per female per spawn) and has a relatively short generation time of 2-4 months. Because foetal development in the transparent egg can be followed in a petri dish, the fish is also a valuable resource in biological research. 

All details – even each individual cell division – in the early development of the fertilized egg can be observed under the microscope. The tiny fish larva hatches just three days after fertilization. The larva is also transparent, which permits microscopic analysis of all its organ. Researchers have access to many thousands of diferent variants and mutants with different attributes for detailed study. The transparent zebrafish (Figure 1) is especially useful for investigating internal reactions in adult fish too. The zebrafish complete genome is well known which is a huge advantage for researchers in their efforts to decode gene regulation in higher animals.

Figure 2. The zebrafish Danio rerio, an Indian striped tropical fish, has in recent years become popular with researchers conducting animal experiments. Photo: Louise von Gersdorff Jørgensen

Behavioural measurements

A zebrafish reacts rapidly to changes in its environment. With its innumerable sensory cells, the fish relays a message from its skin, fins, gills, lateral line (sensory) system, eyes, oral cavity and nares to its brain, which responds by activating the body's peripheral nerves and muscles. Te resulting pattern of movement in the fsh may be recorded in various ways, but with the aid of a video-based computer system, the researcher can set up reproducible behavioural measurements. The fish is placed in a small tank, and its movements are recorded by two video cameras hooked up to a computer (Figure 3).

A literary favourite

Within the last decade especially, the species has risen to unprecedented fame in the international scientific community. From 1990 to 2015, thousands of research articles have been published on zebrafish. Advances in this field of research have been impressive in recent years. This was clearly reflected at the European Zebrafish Meeting this year, which convened more than 800 researchers on the topic of the zebrafish as a model. 

The zebrafish is currently aiding research related to the environment, nutrition, human growth, immunity, cancer biology, genetics, cardiac function, diabetes, lifestyle disease, inflammatory bowel disease and pharmaceutical products, to mention but a few of the most popular felds of research.

Because the fish is so easy to keep, it is also used as a model for farmed fish (carp, trout, salmon, seabream, European seabass), which is invaluable for fish farmers, who supply food fish for the planet's population.

Organ functions and interaction

The zebrafish is equipped with a diverse abundance of organs with glandular functions, as seen in higher animal species. Te organs are not always as welldelimited as seen in mammals, and its pancreatic and thyroid tissue is more diffusely arranged along its digestive tract. The kidney is well-delimited, while its adrenal tissue is dispersed as interrenal cells in the anterior portion of its kidney. 

Hormonal production in the tissue makes it possible to characterise the location and extent of the organs. By using specifc antibodies reacting with insulin it is possible to gain insights into the state of the insulin-producing beta cells in the pancreas. Specifc antibodies against the hormone thyroxin bind to cells in the thyroid, just as antibodies against the stress hormone cortisol bind to the interrenal cells. In the same way, sex hormones in the ovaries and testes can be localized and provide information about reproductive dependency on the body's general condition and interaction with other organ systems.

Figure 3. Zebrafish behaviour may be observed in different ways. Here a system is being configured and calibrated for video recording and computer analysis of zebrafish movement. Photo: Kurt Buchmann

Immune response

The fish's immune system bears many resemblances to that of human beings, and this is also the reason why the zebrafish has become so popular for modelling the human body's reaction to infectious diseases. The zebrafish's ancestors arose 450 million years ago, and, from this prototype, developed into amphibians, reptiles, birds and mammals. All of these animals possess an adaptive immune system; the system which gives humans the ability to recall and react specifcally to certain pathogens (diseasecausing germs). This is the reason why we are now able to vaccinate our children against a large number of diseases. 

The immune system's main organs in the zebrafish comprise the anterior part of the kidney, the so-called head kidney; the thymus located in the gill cavity and the spleen (in the abdominal cavity). This permits researchers to follow how immune system stem cells from the head kidney mature in the thymus when their fate is to end life as T cells. 

The spleen is seen to have both B and T cells, which together with macrophages deal effectively with any pathogens they come across. 

Brain activity

Sensory impressions are transmitted from the surface of the zebrafish to its central nervous system, where impressions are processed and stored and reactions triggered. Environmental impacts are read as a change in the fish's behaviour, but in advance of the reaction, the process involves a series of biochemical processes in the fish's nervous system. Although the zebrafsh nervous system is a good deal less complex than it is in humans, it does allow us to describe a number of basic functions in the central nervous system. Communication proceeds by means of nerve signalling substances known as neurotransmitters. One of those signalling substances is serotonin, which can now be observed in the zebrafish brain during OPT (Optical Projection Tomography) scanning. This technique is based on the knowledge that specifc antibodies are able to bind to the transmitter molecule in special sections of the brain.  

Signalling molecules

As far back as 450 million years ago, the early ancestors of the zebrafish possessed basic immune system elements such as B lymphocytes, T lymphocytes, dendritic cells, macrophages and granulocytes. These immune system elements were capable of producing specifc antibodies which, combined with a number of other proteins, kept the body free of infections. 

The responses are guided and controlled by means of a number of cell-signalling molecules, cytokines, which ensure the smooth functioning of a complex network of reactions. Based on established knowledge of the zebrafsh genome, we have developed a toolbox. We use this to shed light on how more than 45 genes for immune system molecules are activated in a coordinated sequence when the zebrafish is exposed to an environmental impact or infection.  

The transparent zebrafish allows us to demonstrate how pathogenic bacteria are absorbed internally via its surfaces (skin, fins and intestine). By using light-emitting bacteria (Figure 4), the investigator can observe how particles and even dead bacteria find their way from the environment into the inside of the fish, where defence reactions are immediately initiated with the purpose of eliminating the foreign substances. 

Figure 4. The zebrafish is regarded as an integrated organism that triggers a whole series of immunological functions in response to environmental changes. Here fluorescent bacteria are absorbed through the fish's surface. Photo: Louise von Gersdorff Jørgensen

Environmental impact

The zebrafish's functions in its many organs are closely linked. When the fish is exposed to an adverse environmental impact, a stress hormone is produced in the head kidney's interrenal cells. Cortisol, as this stress factor is called, then rushes into the blood stream and bonds with immune cells and organs with the result that resistance is impaired and the invasive bacteria are unhindered in their attempt to debilitate the fish. The fish's basic physiological functions are impaired and the fish's behaviour changes. 

Conversely, the zebrafish can also be used to demonstrate how this physiological network is optimised when the fish is stimulated by other factors. Immune-system stimulants optimise the fish's immunological reactions to improve its state of health. 

In this way, the zebrafish and our linked system of research tools serve as a flexible model of the complex network of organs, signalling molecules and effector molecules that also occur in higher animals, including human beings. This includes the brain, the peripheral nervous system, the heart, liver, kidney and spleen and, not least, the immune system's amazing responses. From being a cute pet in a home fish tank, the zebrafish has gone on to become an experimental star, blazing a trail for a better environment and improved aetiologies (understanding of how disease is caused).