Animals in research: zebrafish

Zebrafish are probably not the first creatures that come to mind when it comes to animals that are valuable for medical research.

You might struggle to imagine you have much in common with this small tropical freshwater fish, though you may be inclined to keep a few “zebra danios” in your home aquarium, given they are hardy, undemanding animals that cost only a few dollars each.

Yet each year more and more scientists are turning to zebrafish to unravel the mechanisms underlying their favourite genetic or infectious disease, be it muscular dystrophy, schizophrenia, tuberculosis or cancer.

My (conservative) estimate is that zebrafish research is now carried out in at least 600 labs worldwide, including 20 in Australia.

So what is it about zebrafish that has taken them from the freshwater rivers and streams of Southeast Asia, beyond the pet shops and into universities and research institutes the world over?

A short history of zebrafish

A scientist called George Streisinger, working at the University of Oregon in Eugene, USA in the 1970s and 80s, recognised the vast potential of this organism for developmental biology and genetics research.

In contrast to fruit flies and worms, the other simple model organisms established at the time, zebrafish are vertebrates.

They have a backbone, brain and spinal cord as well as several other organs, including a heart, liver and pancreas, kidneys, bones and cartilage, which makes them much more similar to humans than you may have otherwise thought.

But as a vertebrate model, could they be as useful as mice?

Several things captured Streisinger’s imagination.

Most famously, zebrafish embryos, unlike mouse embryos, develop outside the mother’s body and are transparent throughout the first few days of life.

This provides unparallelled opportunities for researchers to scrutinise the fine details of embryonic vertebrate development without first having to resort to invasive procedures or killing the mother.

But this advantage is enhanced by the fact zebrafish reproduce profusely (each pair can produce 200-300 fertilised eggs every week); an ideal attribute for genetic studies. Again, the large, external embryos are a critical part of this success.

When just one or two cells old, zebrafish embryos can be easily microinjected with mRNA or DNA corresponding to genes of interest; undeterred, they then they go on to grow and reproduce, handing down the injected gene to the next generation.

From zebrafish to humans

A paper published last month in Nature unveiled the long-awaited sequence of the zebrafish genome, revealing that zebrafish, mice and human have 12,719 genes in common.

Put another way, 70% of human genes are found in zebrafish.

But even more notable is the finding that 84% of human disease-causing genes are found in zebrafish.

Perhaps not surprisingly then, when these genes are injected into zebrafish embryos, the growing animals are doomed to acquire the same diseases.

And while zebrafish are still used widely to answer fundamental questions of developmental biology, much current research is directed towards combining their many attributes in studies that are designed to improve human health.

This is especially true for cancer research where the expression of cancer-causing genes (oncogenes) can be directed to specific organs, virtually at will.

This process, known as transgenesis, is very straightforward in zebrafish and has allowed researchers to produce zebrafish models of liver, pancreatic, skeletal muscle, blood and skin cancers, to name but a few.

And when the genomic make-up of these zebrafish tumours is deciphered using the latest DNA sequencing technology, the patterns of mutations, or “gene signatures”, are found to overlap substantially with those in the corresponding human tumours.

Trialling cancer drugs

These parallels have encouraged researchers to exploit zebrafish in drug development – in particular for high throughput approaches such as chemical/small molecule screens.

Here, the ability to generate tens of thousands of zebrafish embryos harbouring the same disease-causing mutations is crucial.

Then, as the tumours grow in the synchronously developing larvae, the fish are transferred to small volumes of water containing chemicals that may stop the growth, or better still, kill the cancer cells.

Large collections of drugs can be screened relatively quickly for anti-cancer efficacy in this way.

One drug, Leflunomide, identified in such a screen is now in early phase clinical trials to kill melanoma cells.

The only other drug from a zebrafish chemical screen currently in clinical trials is dimethyl-prostaglandin E2 (dmPGE2).

There, the intent is not to kill cancer cells but rather to make mainstream leukaemia treatment more effective.

Studies of dmPGE2 increased the number of blood stem cells in zebrafish embryos and it is being trialled now as a way to expand the number of stem cells in human cord blood samples.

Human cord blood samples are a valuable commodity to restore bone marrow in leukaemia patients after high dose chemotherapy when a matched bone marrow transplant is unavailable.

But the success of this approach is currently limited by the scant number of stem cells in individual cord blood samples, requiring the use of two precious samples for each patient.

Tumour growth

As well as the transgenic zebrafish models of cancer described above, researchers are also transplanting cells derived from human tumours into zebrafish embryos and watching them grow and spread.

The creation of a transparent (non-striped) version of adult zebrafish (called casper, after the cartoon ghost) means the behaviour of tumour cells inside these living organisms can be followed for days at a time.

Coupled with the advent of high resolution live-imaging techniques, the birth, growth and spread of tumours can be scrutinised in movies that can be played over and over again.

These experiments are usually conducted in zebrafish that have been genetically modified to express genes that glow in specific body compartments, giving researchers the ability to pinpoint potentially critical connections between “host” cells and tumour cells that may determine whether the latter survive or die.

This type of experiment is revealing a complex interplay of potentially beneficial and detrimental components.

While the proximity of immune cells may instigate mechanisms capable of destroying the tumour, the stimulation of new blood and lymphatic vessel growth towards the tumour is more insidious, since it delivers the tumour with both the nutrients it needs to survive and a network to spread throughout the body.

These processes, once properly understood, are likely to provide opportunities for therapeutic intervention in the future.

The future of zebrafish

Cancer research is just one part of the zebrafish story. In Australia alone, investigators are also using zebrafish to study metabolic disorders such as:

• diabetes
• muscle diseases, including muscular dystrophy
• neurodegenerative disease
• the response of the host innate immune system to bacterial and fungal infections

Excitingly, research is also underway in this country to unravel the genetic mechanisms controlling heart, skeletal muscle and nervous tissue regeneration in zebrafish, in the hope that these processes can be one day recapitulated in humans to address the burgeoning socioeconomic problem of tissue degeneration in our ageing population.

So next time you peer into someone’s home aquarium, imagine the biomedical possibilities inherent in this lively and amiable little fish!

Zebrafish Genome Found Strikingly Similar to Humans

According to a paper published in Nature, 70 per cent of protein-coding human genes are related to genes found in the zebrafish (Danio rerio), and 84 per cent of genes known to be associated with human disease have a zebrafish counterpart.

The team developed a high-quality annotated zebrafish genome sequence to compare with the human reference genome. Only two other large genomes have been sequenced to this high standard: the human genome and the mouse genome. The completed zebrafish genome will be an essential resource that drives the study of gene function and disease in people.

Zebrafish are remarkably biologically similar to people and share the majority of the same genes as humans, making them an important model for understanding how genes work in health and disease.

“Our aim with this project, like with all biomedical research, is to improve human health. This 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,” said study senior author Dr Derek Stemple of the Wellcome Trust Sanger Institute.

Zebrafish research has already led to biological advances in cancer and heart disease research, and is advancing our understanding of muscle and organ development. Zebrafish have been used to verify the causal gene in muscular dystrophy disorders and also to understand the evolution and formation of melanomas or skin cancers.

“The vast majority of human genes have counterparts in the zebrafish, especially genes related to human disease. This high quality genome is testament to the many scientists who worked on this project and will spur biological research for years to come. By modeling these human disease genes in zebrafish, we hope that resources worldwide will produce important biological information regarding the function of these genes and possibly find new targets for drug development,” explained senior author Prof Jane Rogers, also of the Wellcome Trust Sanger Institute.

The zebrafish genome has some unique features, not seen in other vertebrates. They have the highest repeat content in their genome sequences so far reported in any vertebrate species: almost twice as much as seen in their closest relative, the common carp. Also unique to the zebrafish, the team identified chromosomal regions that influence sex determination.

The zebrafish genome contains few pseudogenes – genes thought to have lost their function through evolution – compared to the human genome.

The team identified 154 pseudogenes in the zebrafish genome, a fraction of the 13,000 or so pseudogenes found in the human genome.

“To realize the benefits the zebrafish can make to human health, we need to understand the genome in its entirety – both the similarities to the human genome and the differences. Armed with the zebrafish genome, we can now better understand how changes to our genomes result in disease,” said Prof Christiane Nüsslein-Volhard, co-author and Nobel laureate from the Max Planck Institute for Developmental Biology.

“This genome will help to uncover the biological processes responsible for common and rare disease and opens up exciting new avenues for disease screening and drug development,” Dr Stemple said.

First steps of synapse building captured in live zebra fish embryos

Using spinning disk microscopy on barely day-old zebra fish embryos, University of Oregon scientists have gained a new window on how synapse-building components move to worksites in the central nervous system.

What researchers captured in these see-through embryos — in what may be one of the first views of early glutamate-driven synapse formation in a living vertebrate — were orderly movements of protein-carrying packets along axons to a specific site where a synapse would be formed.

Washbourne addresses:

► The basic importance of the findings

► The connection to diseases, including autism

The discovery, in research funded by the National Institutes of Health, is described in a paper placed online ahead of publication in the April 25 issue of the open-access journal Cell Reports. It is noteworthy because most synapses formed in vertebrates use glutamate as a neurotransmitter, and breakdowns in the process have been tied to conditions such as autism, schizophrenia and mental retardation.

The zebra fish has become one of the leading research models for studying early development, in general, and human-disease states.

In this case, researchers used immunofluorescence labeling to highlight the area they put under the microscopes. The embryos they studied were barely 24-hours old and a millimeter in length, but neurons in their spinal cord were already forming connections called synapses. Images were taken every 30 seconds over two hours.

"If we zoom out a bit and look at development in the human, the majority of synapse formation occurs in the cortex after birth and continues for the first two years in a baby’s life," said Philip Washbourne, a professor of biology and member of the UO’s Institute of Neuroscience.

Previous studies, done in vitro, contradicted each other, with one, in 2000, identifying a single packet of building blocks arriving at a pre-synaptic terminal. The other, in 2004, identified two protein packets. After watching the process unfold live, with imaging over long time spans, Washbourne said: “We now see at least three, and maybe more, such deliveries.”

"Axons are long processes — think of them as highways — of neurons. In humans, these can be a meter long, from spinal cord to your big toe," he said. It’s in the cell body where all the proteins are made, and they have to be transported out. Is it done by a single bus or by several cars? These results point to additional layers of complexity in the established mechanisms of synaptogenesis."

The new research also showed that sequence also is crucial. Two different pre-synaptic packages of molecules repeatedly arrived in the same order. A key building block — the protein synapsin — always arrived third. As these delivery vehicles traveled the axonal highway, another protein, a cyclin-dependent kinase known as Cdk5, acts as a stoplight at the synapse-construction site, where phosphorylation occurs. More research is needed on Cdk5, Washbourne said.

"Understanding how all this happens will inform us to what’s going wrong in neurodevelopment that leads to diseases," Washbourne said. "We have indications that the glue that gets all this going includes a gene that has been linked to autism, so knowing how these molecules start the process of synapse formation — and what goes wrong in people with mutations in these genes — might allow for a therapeutic targeting to correct the mutations and manipulate the stop signs."

Researchers image most of vertebrae brain at single cell level

Misha Ahrens and Philipp Keller, researchers with the Howard Hughes Medical Institute have succeeded in making a near real-time video of most of a zebrafish’s brain showing individual neuron cells firing. To create the video, as the team reports in their paper published in the journal Nature Methods, the two developed a type of modified light-sheet microscopy and used it in on genetically modified fish.

To create the video, the researchers turned to zebrafish in their larval state—their brains are transparent and small. To cause firing neurons to be visible they genetically altered the fish’s brains, giving them a protein that glows when responding to changes in calcium ion levels, which happen when nerve cells fire. Next, they used a microscope that was able to broadcast a sheet of light through the fish’s brain allowing for the detection of the firing neurons. The system recorded images every 1.3 seconds. The final step was stitching the images together to create a video. The result is nothing short of breathtaking—looking like something out of a science fiction movie’s special effects department.

The video marks the first visual capture of most of a living vertebrae brain at the neuron level, as it works in near real-time and offers striking evidence of the complexity of the brain—even one as small as 100,000 neurons. The researchers say their video shows approximately 80 percent of the zebrafish’s brain as it operates—though what all those firing neurons represent in particular, is still unknown.

The researchers are careful to point out that what they’ve accomplished does not portend the creation of a video of a human brain in action—our brains are much larger, have billions more neurons and perhaps more importantly, are not transparent and are covered by a thick skull. Instead they suggest that studying a simpler brain in action might help to explain how biological neural networks actually work, perhaps leading to theories that can be generalized over larger animals.

But before that can happen, the procedure the team has developed needs to be improved—neurons can fire at hundreds of times per second, which means a lot of firing in the video has been missed. Capturing at a faster rate would mean generating nearly unmanageable amounts of data—at the current rate, just one hour of capture creates a terabyte of data. Thus a new way to store and process the data must be developed.

Research update: Imaging fish in 3-D

Zebrafish larvae — tiny, transparent and fast-growing vertebrates — are widely used to study development and disease. However, visually examining the larvae for variations caused by drugs or genetic mutations is an imprecise, painstaking and time-consuming process.

Engineers at MIT have now built an automated system that can rapidly produce 3-D, micron-resolution images of thousands of zebrafish larvae and precisely analyze their physical traits. The system, described in the Feb. 12 edition of Nature Communications, offers a comprehensive view of how potential drugs affect vertebrates, says Mehmet Fatih Yanik, senior author of the paper.

“Complex processes involving organs cannot be accurately recapitulated in cell culture today. Existing 3-D tissue models are still far too simple to model live animals,” says Yanik, an MIT associate professor of electrical engineering and computer science and biological engineering. “In whole animals, the biology is far more complicated.”

Lead authors of the paper are MIT graduate student Carlos Pardo-Martin and Amin Allalou, a visiting student at MIT. Other authors are MIT senior research scientist Peter Eimon, MIT intern Jaime Medina, and Carolina Wahlby of the Broad Institute.

Zebrafish are genetically similar to humans and have many of the same developmental pathways, so scientists often use them to model human diseases including cancer, diabetes, Parkinson’s disease and autism.

Using the new technology, researchers can grow larvae in tiny wells and flow them through a channel to an imaging platform. Once there, the embryos are rotated and 320 images are taken from different angles, allowing 3-D reconstructions to be made using optical projection tomography (OPT). Getting larvae to the platform takes about 15 seconds, and the imaging takes only 2.5 seconds. This allows hundreds or thousands of larvae to be imaged within hours.

In a 2010 paper, Yanik’s team described the system that transports the embryos to the imaging platform, which they combined with high-resolution two-dimensional imaging. In the latest version, they developed a high-speed OPT imaging technique, which takes hundreds of two-dimensional images and subsequently generates a 3-D image, similar to a CT scan.

They also created a computer algorithm that can measure hundreds of traits and use that information to create a comprehensive phenotype map — the overall description of an organism’s characteristics — for each larva. This enables rapid and detailed studies of how different drugs affect those phenotypes.

“You could probably look at almost any organ or tissue that you’re interested in,” Eimon says. “It gives researchers a way to rapidly measure and quantify and put numbers on the kinds of phenotypes and gene-expression patterns that they’ve been looking at for years and years.”

In this study, the researchers focused on the craniofacial skeleton, which is analogous to the human skull. They measured the length and volume of each of the bones that make up this structure, as well as the angles between the bones.

Each embryo was imaged five days after being treated with one of nine different teratogens — drugs that cause developmental abnormalities. The researchers compared their results with the drugs’ known effects and found that they were very consistent. They also obtained high-resolution, 3-D images of the craniofacial skeletons, which are less than a millimeter long.

“Now that we’re able to load the animals, and we can image them really quickly, and we have a way to start looking at the information, the sky’s the limit,” Pardo-Martin says. “What we have to do now is ask the big questions, because the technology has advanced.”

This kind of analysis could be very valuable for drug developers who need to efficiently screen thousands of drug candidates. It could also be used to study hard-to-detect changes in phenotype caused by genetic mutations, says Joseph Fetcho, a professor of neurobiology and behavior at Cornell University.

“A really high-throughput way to assess phenotype is very important for measuring small effects on the development of an organism,” says Fetcho, who was not part of the research team. “You can see what the phenotype looks like in a large population and quantify it in a very rigorous way.”

The zebrafish revealed a central regulator for the development of the brain histamine system

Research has shown that mutations in the psen1 gene are common in the familial forms of Alzheimer’s disease, and the Presenilin-1 protein that the gene encodes is known to be involved in the cleavage of the amyloid precursor protein. In Alzheimer’s disease the amyloid precursor protein is not cleaved the normal way, and the protein accumulates in the brain damaging neuronal tracts and neurons. It is still unknown if the psen1 gene is involved in the etiology of Alzheimer’s disease via another mechanism.

Professor Pertti Panula’s research team at the University of Helsinki has elucidated the role of psen1 gene in the development of the neuronal histamine system and its modulation. Histamine is one of the neurotransmitters, which all are essential for cognitive functions, which in turn are impaired in Alzheimer’s disease. The histamine system is altered during the progression of Alzheimer’s disease.

In the study the zebrafish was used as a model organism. The rapidly developing zebrafish is suitable as a model organism, as its transparency allows researchers to study the development and function of vital organs. To study the function of psen1 gene, zebrafish that did not produce functional Presenilin-1 protein were generated. Despite the fact that the fish lacked functional Presenilin-1 they were viable and developed until adulthood.

The lack of Presenilin-1 protein induced a change in the behavior of the larval zebrafish, they did not as normal fish react to fast changes in the light intensity. “Based on previous research we know that this change in behavior is associated with lack of histamine in the brain”, Panula explains.

In adulthood the motor behavior of the mutant zebrafish differed from the normal fish: the fish swam by the edges of the arena that was available and avoided the inner part. Previous studies from the group have shown that this behavioral alteration also is due to changes in the histamine system.

The researchers found that larval fish lacking Presenilin-1 protein had significantly fewer histamine neurons; in adulthood the histamine neuron number was significantly increased in these fish when compared with normal fish.

"These results reveal that the psen1 gene is a central regulator of the development of the histamine neurons and that the mutation can cause a persistent lifelong change in the neuronal histamine system. This is a very interesting finding", Panula states.

One interesting remaining question is from where the new histamine neurons arise in the brains of adult zebrafish. Are they newly differentiated stem cells or do other cells become histamine neurons? The answer is not known, but based on these results it is advisable to elucidate the role of Presenilin-1 protein in differentiation of stem cells also in the brains of mammals. “Mammals have stem cells in the hypothalamus, in the same area where the histamine neurons are located in all studied vertebrates”, Panula comments.

Panula empathizes that the published study does not tell about an Alzheimer’s disease mechanism in humans. The new knowledge on the function of psen1 gene and the development of the brain histamine system provided by the study is one step forward to understanding the etiology of the disease.

"We perform basic research on molecular level, from where it is a long way to treatment of human diseases. This type of research provides the findings on which the treatments are finally based", Panula says.

Journal of Neuroscience published the study that was conducted at University of Helsinki Neuroscience center, and Institute of Biomedicine.

(Image: Charles Badland, Florida State University)

What’s Your Fish Thinking?

Studying the links between brain and behavior may have just gotten easier. For the first time, neuroscientists have found a way to watch neurons fire in an independently moving animal. Though the study was done in fish, it may hold clues to how the human brain works.

"This technique will really help us understand how we make sense of the world and why we behave the way we do," says Martin Meyer, a neuroscientist at King’s College London who was not involved in the work.

The study was carried out in zebrafish, a popular animal model because they’re small and easy to breed. More important, zebrafish larvae are transparent, which gives scientists an advantage in identifying the neural circuits that make them tick. Yet, under a typical optical microscope, neurons that are active and firing look much the same as their quieter counterparts. To see what neurons are active and when, neuroscientists have therefore developed a variety of indicators and dyes. For example, when a neuron fires, it is flooded with calcium ions, which can cause some of the dyes to light up.

Still, the approach has limitations. Traditionally, Meyer explains, researchers would immobilize the head or entire body of a zebrafish larvae so that they could get a clearer picture of what was happening inside the brain. Even so, it was difficult to interpret neural activity for just a few neurons and over a short period of time. Researchers needed a better way to study the zebrafish brain in real time.

Enter Junichi Nakai of Saitama University’s Brain Science Institute in Japan. He and colleagues selected a glowing marker known as green fluorescent protein (GFP) and linked it to a compound that would light up in the presence of large amounts of calcium. The researchers then inserted the DNA that codes for this marker into the zebrafish genome, tying it to a specific protein only found in neurons. This means that only actively firing neurons would fluoresce, and scientists could track neural activity without applying dye. Because the signal was stronger and clearer, researchers didn’t have to immobilize the larvae.

To test the setup, Nakai and colleagues sent the genetically engineered zebrafish larvae hunting for food. When the larvae see a swimming single-celled animal called a paramecium, they engage in what animal behaviorists call a prey capture response: They turn their heads toward the paramecium, swim at it, and finally eat it.

Using their newly developed imaging system, Nakai and colleagues associated the sight of moving paramecium and prey capture behavior with the activation of a group of neurons in the optic tectum, the visual center of the zebrafish brain. The neurons pulsed in tandem with the movements of the paramecium—a sudden dart of the one-celled organism caused a bright flash of neural activity in the zebrafish tectum. The tectum went silent if the paramecium stilled. Only moving prey interested the larvae, the team reports today in Current Biology. These particular neurons, Nakai proposes, are part of a specific visual-motor pathway that links the sight of moving prey with swimming behavior.

"It’s a good proof of principle study," Meyer says. "The most important thing is that they showed [the technique worked] on freely behaving fish."

Genetic Researchers Grow A Fish That Has Legs

The fossil record has a lot of strange stories to tell about the evolution of life on Earth, and one of the strangest is how life moved from sea to land. Though clues from the record give the rough outlines of the story—limbs grew from fins in a series of stages in which fins grew longer and narrower—scientists are still filling in the details, trying to determine what genetic changes might have allowed the limbs to grow.

One of the best ways to learn those details is to reproduce the changes that occurred some 400 million years ago—to virtually back in time and alter the development of the land-goer’s living ancestors and see what happens.

Which is what biologist Renata Freitas and colleagues were up to when they added some extra Hoxd13—a gene known to play a role in distinguishing body parts during embryological development— to the tip of a zebrafish embryo’s fin, and watched as the developing fin kept growing.

Their lab findings led the researchers to hypothesize that the secret to limb development may have been a new element in some lobe-finned fish’s DNA. When present, this DNA element would have helped turn on the Hoxd13 gene on the fish embryo’s fins, leading them to lengthen and grow into limbs.