WCD2016 Special Feature: The lab’s next top model

These little swimmers are capturing the hearts of researchers as model organisms for various scientific studies

By: Syamil Zahari

Think of the common research animals, and your mind might picture various typical lab residents: rats, mice, fruit flies perhaps, or maybe even worms.

Over the recent decades, however, one species is increasingly becoming scientists’ favourite biomedical laboratory bench buddies, especially in the human genetic disease research such as cancer studies.

Danio rerio, or zebrafish, swam into view in the early 1980s when scientists started to recognize its vast potential in the field of developmental biology and genetics, subsequently becoming one of the chosen few from the animal kingdom – humanity included – to have been flown into space.

Zebrafish is a tropical freshwater fish, about 2.5-inch in length with horizontal stripes across its body (hence the moniker), belonging to the minnow family that is native to the streams and ponds of the Himalayan region. An aquarium favourite worldwide, it is a common stock in the trade.

In research labs, however, the species have distinct biological advantages. Unlike fruit-flies, worms or other small models, zebrafish are vertebrate animals. They have a backbone, brain and spinal cord as well as organs such as heart, liver, pancreas, kidneys, bones, cartilage and others, rendering them ideal test subjects for human diseases.

Furthermore, the zebrafish genome has already been fully sequenced – it shares 70% of its genetic code and 84% of disease-causing genes with human – making zebrafish suitable testable for developmental behaviors.

Small fish, big splash

Recognizing these advantages, Cancer Research Malaysia’s Drug Discovery Team is one of the growing number of laboratories globally that has been utilizing zebrafish. Cancer Research Malaysia, which aims to tap into Malaysia’s wealth of biodiversity for potential anti-cancer drugs, is using zebrafish phenotypic assay as multi-pathway screening model.

“Zebrafish phenotypic assay is one way to discover new target-specific anti-cancer compounds from natural products,” according to the organization.

“Since embryogenesis and carcinogenesis have common critical pathways, a chemical that interferes with embryogenesis could also affect the development of cancer in a pathway-specific manner,” it stated.

This is due to the fact that zebrafish embryos and larvae are relatively large and initially transparent, hence allowing for in vivo imaging with a simple microscope, “thereby allowing easy observation of their development and any defects arising from exposure to natural products,” according to Cancer Research Malaysia.

The Drug Discovery team aims to scrutinize the targeted signalling pathways involved in zebrafish embryogenesis and human carcinogenesis, as well as inhibitors of tumor angiogenesis, to identify potential drug candidates.

“This is made possible by screening natural products for the appearance of abnormal phenotypes in developing zebrafish embryo which if observed that this is an indication of chemical disruption on specific embryo developmental genes in zebrafish.”

Zebrafish embryos are also permeable to many chemicals and drugs, which make them ideal for screening large numbers of toxicology samples. Moreover, zebrafish embryos develop ex utero, i.e. outside the mother’s body, and the eggs will remain translucent for up to five days. This provides unique opportunities for researchers to scrutinize the finer details of embryonic development without either resorting to invasive procedures or killing the mother.

The embryos are also easily manipulated by microinjecting them with tumours cells, allowing scientists using high resolution live-imaging techniques to closely monitor the tumour cells’ growth and spread throughout the development of the embryos.

Zebrafish can be also genetically altered to express genes that glow in specific body compartments, giving researchers the ability to pinpoint potentially critical connections between host cells and tumour cells. Along with the creation of DNA-modified transparent (non-striped) version of adult ‘Casper’ zebrafish, the behaviour of tumour cells inside these living organisms can be examined for days at a time.

Earning their stripes

Dr Kazuhide Shaun Okuda is a postdoctoral scientist with Cancer Research Malaysia’s Drug Discovery team who has been working on zebrafish phenotypic assay project.

“The basic overview of what we do is that we use natural resources in Malaysia to try to identify anti-cancer drugs using the tools that we have. The tool that specifically we are using is this animal model, the zebrafish,” he explained during an exclusive interview with AMOR Media.

“There’re specific things that is faster and better using zebrafish but, of course, the trend is starting to gain momentum. The main selling point is that it’s cheaper and you can see significant results quickly,” Okuda said.

A study has calculated that a tank of zebrafish costs 6.5 cents a day to maintain, compared to 90 cents for five mice in a cage. In addition, unlike rodents, zebrafish can be kept in high densities, even by the thousands, in small tanks. They are also relatively inexpensive to feed and to transport (as embryos) between labs.

Making the species even more economically viable, zebrafish reach maturity quickly and are able to reproduce in great numbers quite often in its three-year lifespan. Adult zebrafish can grow to about two inches in three months, and a pair of zebrafish can produce embryos by the hundreds after only three days of post-fertilization.

The quick turnaround proves valuable to scientists especially in genetic studies. As several generations are typically requisite before a stable modification of the targeted gene is reached, zebrafish’s rapid breeding greatly reduces the time needed. In the research of particularly rare genes, having large numbers of second filial generation (F2) fish can make the process even much faster and more efficient.

Growing opportunity

Last November, to promote opportunities for collaborative research efforts utilizing zebrafish, Cancer Research Malaysia with Dr Okuda as lead organizer put together the 1st Malaysia Zebrafish Disease Model Workshop.

The two-day workshop featured a keynote presentation by Prof Philip Stanley Crosier, Professor of Molecular Medicine in the School of Medical Sciences at the University of Auckland, New Zealand, who is one of the leading scientists with expertise on zebrafish modelling. Prof Crosier’s laboratory has already generated several transgenic zebrafish lines that fluorescently label cellular compartments such as neutrophils and haematopoietic stem cells.

“We know that cancer is a genetic disease, so if you can understand aspects of cancer using a powerful genetic model like the zebrafish, you can begin to pull apart the disease process,” he explained during an interview with Scientific Malaysian magazine.

“Cancer is a complex disease. What I mean by that is that cancer results not only from one gene functioning aberrantly, but it is a collection of genes coordinating in some manner to drive cancer progression,” he said.

“So if you have a highly genetically tractable, manipulatable and transparent model organism, you can then start to visualise cell migration, for example cancer stem cells, in the development of zebrafish,” he added.

“This is a growing opportunity as you can use them as a platform in the drug discovery process. The whole process is scalable, so you can develop biological readouts of these zebrafish systems and start finding small molecules, drugs or even natural product extracts that might work in these targets that you have identified,” Prof Crosier said.

Targeted therapies

With targeted therapies gaining favour as the preferred therapeutic approach in the treatment of cancer, the use of zebrafish’s underlying DNA alteration earns the species a distinctive prominence as an important vertebrate model for investigating various human diseases including cancer.

“Zebrafish provides unique advantages, for example optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it perfect complement to the murine model in biomedical research,” stated Cancer Research Malaysia.

“Recent advances, which includes the generation of novel transgenic lines that enable real time imaging of neuronal activities, the use of cancer cell line or tissue xenotransplantation techniques, and the development of efficient knock-out/knock-in technologies, now allow the detailed investigation of human disease in zebrafish,” the organization added, making the zebrafish model a promising tool in the search for targeted cancer therapeutics from natural products.

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