zebrafish

James Amatruda, MD, PhD, never set out to become a fish farmer.

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“My goal is not to cure a fish; it’s to cure a child,” Amatruda, head of basic and translational research for the Cancer and Blood Disease Institute and Dr. Kenneth O. Williams chair in cancer research at Children’s Hospital Los Angeles, told Healio. “I see the impact these cancers have on children and their families every day, and that spurs to us to go back to the laboratory and work that much harder.”

As it turns out, much of the hard work at Amatruda’s lab involves some incidental fish farming — in fact, his 3,000-square-foot facility is designed to house up to 120,000 zebrafish. The cancer researcher chose the zebrafish model for its anatomical similarity to humans, a fact that may surprise some.

“The more we look at it, the more we realize just how similar humans and zebrafish are,” Amatruda said. “About 80% of human genes have a direct equivalent in the zebrafish and when we look at disease-causing genes, the similarities are even greater.”

Amatruda spoke with Healio about the value of zebrafish models in understanding pediatric cancers, the major progress that has been made in treating these cancers, and the potential future implications of his research.

Healio: You have said that progress in treating pediatric solid tumor cancers has hit a plateau. Why do you think that is?

Amatruda: Through many careful studies and clinical trials and the introduction of new chemotherapy drugs and combinations of chemotherapy, radiation and surgery, the survival rates and outcomes for these solid tumors in children have improved dramatically. However, we’re getting to the point where we can’t increase the intensity of those therapies much more because of concerns about toxicity and side effects. These are short- and long-term effects that will impact the quality of life of children who have survived cancer. So, although we’re very grateful to be able to apply these therapies, we really need to do better in curing patients and minimizing toxicity in tumors that can be treated effectively with chemotherapy.

Healio: What makes zebrafish such a good model for studying the development of tumors during childhood?

Amatruda: Cancers in children are often related to problems during fetal development of organs such as the kidney or liver, or bone or muscle. Under normal circumstances, that growing tissue is supposed to slow down and start taking on the mature function of a muscle cell, or kidney or liver cells. In childhood cancers, very often that process is somehow disrupted, which can lead to a tumor. By studying those mechanisms even in normal development, we can gain incredible insight into how cancers develop. That’s really what led us to using the zebrafish as a model system, because that experimental model organism is extremely well-suited to studying the process of normal development. We can literally start with the first cell and watch that development happen. We can mimic what we think are the causes of childhood cancer and watch those same types of tumors develop in the fish. The zebrafish is very similar to humans in terms of its anatomy, physiology and disease susceptibility. The zebrafish can be models not only of cancer, but also of diabetes, neurodegeneration, inflammatory diseases, infections, skeletal problems and cardiovascular defects.

Healio: Can you describe the work you have done on fusion oncogenes?

Amatruda: Fusion genes is the term we use when two pieces of different DNA chromosomes break apart and then fuse together. When that happens, very often an abnormal protein is made, one that’s never present in normal cells but combines parts of two different proteins. It becomes a new protein that can act as a cancer driver gene. These occur both in leukemia and solid tumors. We have been studying the ones that occur in sarcomas. In the past, we’ve done some work on rhabdomyosarcoma, which is caused by the PAX3-FOXO1 fusion gene. In that case, we used the zebrafish system to learn how those tumors develop. More recently, we focused on Ewing sarcoma, which is the tissue often found in bone and sometimes in soft tissue. This is caused by the EWS-FLI1 fusion oncogene.

EWS-FLI1 was discovered about 30 years ago and, unfortunately, we still don’t have a very specific treatment that targets it. We discovered that, under certain conditions, introducing the EWS-FLI1 gene into zebrafish will cause them to develop a tumor that is strikingly similar to human Ewing sarcoma. So, that’s a very exciting advance.

We’re using this system now for several purposes; one is to study how these tumors developed, starting at a single-cell level all the way up to a fully formed tumor. We are also looking at how the tumor interacts with surrounding nontumor cells, and we can use this system to test new potential therapies. The fish are small, they grow quickly, and we have the potential to grow hundreds of thousands of fish. We are fortunate to work not only within our own lab, but as part of a large international cooperative group trying to attack this problem. Now, we can bring to the table this unique animal model that mimics so many features of the human disease.

We’re working with collaborators, for example, at The University of Texas Southwestern Medical Center in Dallas, where they are searching large chemical laboratories for potential inhibitors of EWS-FLI1. Our goal is to apply those inhibitors to the fish model to validate whether it has an impact on the tumors.

Healio: What is next in your research with this system?

Amatruda: I think we are almost at the starting line of using this model to drive progress in the field. We’re taking on several parallel tracks, one of which is screening any available drugs that might be effective therapies for Ewing sarcoma.

We also now have a unique chance to look at how the immune system might affect the growth and spread of Ewing sarcoma tumors. Immuno-oncology has been incredibly important in childhood leukemia and in lung cancer, melanoma and some other tumors. It’s never worked very well for Ewing sarcoma or other cancers, and we don’t know enough about why that is. So, we are quite dedicated to using this model to study the impact of the immune system on growth of the tumors.

Lastly, one of the biggest problems we face in the clinic is taking care of children in whom the tumor has metastasized to other tissues. Those tumors are very difficult to treat and do not respond well to our current therapy. We are thinking about ways we can use the zebrafish system to study how it is that the metastatic cell is able to break away from the main tumor, move to a different part of the body, and “set up shop” to grow a secondary tumor there. We’d like to understand how that process occurs so we can design better treatments to target it. The fish system enables us to study very directly the interaction of a tumor cell with the cells around it. We also want to study the signaling events that allow the tumor cells to adapt and survive. We think that knowledge may also tell us something about metastasis in other cancer types.

For more information:

James Amatruda, MD, PhD, can be reached at Children’s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027.

Scientists Caught ‘Undead’ Genes Coming Alive After Death.

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What really happens to us after death? Once a person stops breathing, and their heart ceases to pump blood, they’re what doctors consider “clinically dead.” On a biological level, the eventual decomposition of cells, organs, and brain tissue signal its final and irreversible stages.

But what if that’s not actually the end? Two new studies claim that hundreds of genes actually kept expressing—and, in some cases, become more active—after death occurred. This came as a surprise to the researchers, because forensic pathologists have long suspected that gene activity degrades postmortem, which is why their rate of change is sometimes used to calculate time of death.

According to the lead author of both papers, microbiologist Peter Noble of the University of Washington, the discovery of “undead” genes could help to improve the preservation of organs destined for transplantation. The two studies are currently available on the pre-print server bioRxiv, and it’s important to note that neither have undergone peer review yet.

Noble says his most recent research was inspired by a three-year-old study published in Forensic Science International that discovered a host of genes that remained active in human cadavers for up to 12 hours after death.

In order to investigate the unwinding of the genetic clock, in these latest studies, the team extracted and measured messenger RNA (mRNA) levels in the tissue of recently deceased mice and zebrafish. Since mRNA plays an important role in gene expression, higher levels of this molecule should indicate more genetic activity.

In one of the studies, Noble and his colleagues were able to describe more than 1,000 genes that stayed “alive” postmortem. A total of 515 mice genes continued to operate for up to two days, while 548 zebrafish genes remained functional for an entire four days after death.

“It’s an experiment of curiosity to see what happens when you die,” Noble told Science Magazine.

One of the most surprising findings, however, was that hundreds of genes actually fired up—boosting their activity—within the first 24 hours after the animals had died. Noble suspects that many of them might have been suppressed or shut off by a network of other genes when their host was alive, and only after death were they free to “reawaken.”

The team also found that many of the genes that persisted postmortem are typically active during embryonic development, which led them to theorize that, on a cellular level, newly developing lifeforms might share a lot in common with degenerating corpses.

Other genes they identified were associated with promoting the growth of cancerous cells. These researchers believe the activation of cancer-related genes postmortem could partly explain why many transplant recipients are at higher risk of developing cancer after receiving a new organ, although this has long been attributed to the immunosuppressive drugs they’re typically prescribed. A lot more research still needs to be done.

“Since our results show that the system has not reached equilibrium yet,” one of the studies broadly speculates, “it would be interesting to address the following question: what would happen if we arrested the process of dying by providing nutrients and oxygen to tissues? It might be possible for cells to revert back to life or take some interesting path to differentiating into something new or lose differentiation altogether, such as in cancer.”

In addition to offering potentially valuable new insights into the expiration of vital transplant organs, the researchers hope their findings can also be used by forensic scientists to more accurately pinpoint time of death, which is apparently harder than it sounds.

“The headline of this study is that we can probably get a lot of information about life by studying death,” said Noble.

 

Zebrafish discovery boosts stem cell research

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Australian researchers studying zebrafish have made one of the most significant ever discoveries in stem cell research.

They have uncovered the mystery of how a critical type of stem cell found in blood and bone marrow, and essential to replenishing the body’s supply of blood and immune cells, is formed.

The cells, called hematopoietic stem cells (HSC), are already used in transplants for patients with blood cancers such as leukaemia and myeloma.

But HSCs have significant potential to treat a broader range of conditions because they appear to be able to form all kinds of vital cells including muscle, blood vessel and bone.

The problem was scientists had no idea how HSCs formed, making growing them in a lab and using them to treat spinal cord injuries, diabetes and degenerative disorders impossible.

However, a research team led by Professor Peter Currie, from the Australian Regenerative Medicine Institute at Victoria’s Monash University, uncovered a major part of HSC’s development. Understanding how HSCs self-renew to replenish blood cells is considered the holy grail of advancing stem cell research.

Using high-resolution microscopy, Currie’s team filmed HSCs as they formed inside zebrafish embryos. “It’s a sad fact of life that humans are basically just modified fish, and our genomes are virtually identical to theirs,” Currie said. “Zebrafish make HSCs in exactly the same way as humans do, but what’s special about these guys is that their embryos and larvae develop free living and not in utero as they do in humans.

“So not only are these larvae free-swimming, but they are also transparent, so we could see every cell in the body forming, including HSCs.” The researchers were initially studying muscle mutations in the zebrafish. But when playing the film back they noticed that the muscle-deficient zebrafish had several times the normal population of HSCs.

They saw the pre-HSCs required a “buddy” cell, known as endotome cells, to turn into HSCs.

“Endotome cells act like a comfy sofa for pre-HSCs to snuggle into, helping them progress to become fully fledged stem cells,” Currie said.

“Not only did we identify some of the cells and signals required for HSC formation, we also pinpointed the genes required for endotome formation in the first place.

“I’m not an HSC biologist, I’m an muscle cell biologist, so this was a highly serendipitous finding we made because these helper cells are made next to the muscle stem cells we were initially examining.” He said researchers could now focus on finding the signals present in the endotome cells responsible for HSC formation in the embryo.

“Then we can use them in the lab to make different blood cells on demand for all sorts of blood-related disorders,” he said.

If they could do this, there would also be the potential for genetic defects in cells to be corrected and transplanted back into the body, he said.

Their findings were published in the international journal Nature.

Dr. Georgina Hollway, from the Garvan Institute of Medical Research in Sydney, said the work highlighted how molecular processes in the body play a key role in HSC formation.

“We now know that these migratory cells are essential in the formation of HSCs, and we have described some of the molecular processes involved,” Hollway said.

“This information is not the whole solution to creating them in the lab, but it will certainly help.

“It’s difficult to say exactly how close we are, but we have uncovered a vital step in the process.”

 

In vivo cardiac reprogramming contributes to zebrafish heart regeneration.

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Despite current treatment regimens, heart failure remains the leading cause of morbidity and mortality in the developed world due to the limited capacity of adult mammalian ventricular cardiomyocytes to divide and replace ventricular myocardium lost from ischaemia-induced infarct1,2. Hence there is great interest to identify potential cellular sources and strategies to generate new ventricular myocardium3. Past studies have shown that fish and amphibians and early postnatal mammalian ventricular cardiomyocytes can proliferate to help regenerate injured ventricles4, 5, 6; however, recent studies have suggested that additional endogenous cellular sources may contribute to this overall ventricular regeneration3. Here we have developed, in the zebrafish (Danio rerio), a combination of fluorescent reporter transgenes, genetic fate-mapping strategies and a ventricle-specific genetic ablation system to discover that differentiated atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to zebrafish cardiac ventricular regeneration. Using in vivo time-lapse and confocal imaging, we monitored the dynamic cellular events during atrial-to-ventricular cardiomyocyte transdifferentiation to define intermediate cardiac reprogramming stages. We observed that Notch signalling becomes activated in the atrial endocardium following ventricular ablation, and discovered that inhibiting Notch signalling blocked the atrial-to-ventricular transdifferentiation and cardiac regeneration. Overall, these studies not only provide evidence for the plasticity of cardiac lineages during myocardial injury, but more importantly reveal an abundant new potential cardiac resident cellular source for cardiac ventricular regeneration.

Source: Nature

 

 

Animals in research: zebrafish .

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HealthLab_zebrafish_TheConversation

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 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 alsotransplanting 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 thecartoon 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, includingmuscular dystrophy, neurodegenerative disease, and 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!

Source: http://www.sciencealert.com.au

Zebrafish: A Model System to Study Heritable Skin Diseases

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Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ~500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5–6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.

source: nature dermatology