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Tiny Fish Makes A Big Splash In Arizona Medical Research

By Nicholas Gerbis
Published: Friday, February 17, 2017 - 7:01am
Updated: Friday, February 17, 2017 - 12:11pm

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John Collins
(Photo by Nicholas Gerbis - KJZZ)
John Collins, a research associate in Haiyong Han’s lab, maintains TGen’s 90 strains of zebrafish.

In a small room just off the wet labs of the Translational Genomics Research Institute (TGen), there stands a bubbling rack of transparent tanks. Each bears a label listing such information as strain genotype, generation, birthdates and a tank number.

Containers marked “breakfast” and “dinner” stand on a Formica counter just outside the door. Research associate John Collins knows them only too well.

“They’re fed twice with brine shrimp and once with flake food," he said.

This is TGen's zebrafish facility, home to around 700 small creatures. They belong to a species called Danio rerio that has grown, since the late 1960s, into big fish in research circles — in fields from cancer research to genetics development, with stops along the way in heart disease, blood disorders and gut degeneration. They’ve even traveled to space.

And that’s why Collins, who works under Haiyong Han, head of TGen's Pancreatic Cancer Research Unit, spends part of his days maintaining and genotyping Danio rerio strains.

“One of the first projects that we worked on — and that we still work on — was to establish a model for pancreatic cancer in zebrafish,” said Han.

Zebrafish are model species — animals about which scientists know a great deal and that therefore make useful testbeds for understanding disease and development. Small mammals might seem like better stand-ins for humans, but lower vertebrates like zebrafish resemble humans in surprising ways. Beyond the basic organs that vertebrates share in common, Han said that there’s a significant genetic overlap, too.

“For 70 percent of all human genes, we can find a counterpart in the fish," Han said.

Moreover, when scientists find a protein in fish that also occurs in mammals, that protein usually has the same function in both.

But zebrafish also offer practical advantages over other model species. They’re cheap, hardy and require few creature comforts. Most importantly, a single pairing can result in 200-300 eggs, which will hatch within 48-72 hours and reach sexual maturity within 90 days. This rapid development means that any mutation will develop in fast-forward as well.

“We’re able to generate new strains very quickly. And also, for our pancreatic cancer model, we can significantly compress the time it takes for tumors to develop,” Collins said.

Zebrafish also enjoy another clear advantage over other model animals: Their translucent embryos and eggs let researchers study their inner goings-on without killing them. In adults, a tweaked gene can render their characteristic white-and-neon-blue stripes transparent as well.

At TGen, Han’s lab uses zebrafish to understand the development, metastasis and treatment of pancreatic cancer. By introducing mutated K-ras gene, which is associated with pancreatic cancer in humans, and inactivating p53, a tumor-suppressor gene, researchers can cause pancreatic tumors to grow and monitor their progress through the fish’s transparent skin.

Collins, who maintains TGen’s roughly 90 zebrafish strains, said that they can introduce genes during an embryo’s one-cell stage.

“We do that through microinjections. So, that construct can be incorporated into its genome," Collins said. "And that cell will divide, and the cells that make up the organism will contain that gene or construct."

Han’s lab also tests how factors like cholesterol, metabolism and insomnia affect pancreatic cancer — again, by knocking out key genes. Genetic manipulation is difficult, but it beats force feeding Gouda while they binge-watch Shark Week videos. But they don’t just do it for kicks.

“Sometimes, you have certain disorders that are not necessarily going to make your cancer worse — maybe they can prevent cancer formation. That’s possible. That’s why we study different genes involving different things in relation to cancer,” said Han.

Zebrafish embryos also have handy qualities for understanding development and related cellular activity. Vertebrates, be they fish or human, share many cellular development features in common — so much so that some 19th century scientists, like Ernst Haeckel, once believed that animals in the womb went through a sped-up version of evolutionary history.

This Recapitulation Theory has since gone the way of the dodo, but the useful shared developmental patterns that inspired it remain. For Han, they provide a handy way to study treatments for metastasis — the process in which cancer cells break away from a source tumor and form new tumors elsewhere in the body.

The key lies in embryonic stem cells called neural crest cells. As they traverse a developing embryo, en route to differentiate into other cell types, their motion mimics metastasis.

“We label the cells with green fluorescence, and we can monitor the movement of those cells with a microscope. If we treat embryos with a drug, then we can test if the drug can prevent the migration of those cells — and possibly inhibit the metastasis of cancer cells,” said Han.

This relationship lets Han test his treatments: If they can stop neural crest cell migration, perhaps they can stop metastasis.

Elsewhere in TGen, Sampath Rangasamy, a research assistant professor with TGen's Center for Rare Childhood Disorders, uses zebrafish embryos to study epileptic encephalopathies — a diverse group of brain disorders that can cause seizures, brain development deficits and, sometimes, early death in young children.

“You can see infantile patients and epileptic seizures in children as early as 2 months old, 6 months old,” said Rangasamy.

To confirm that a new mutation in the DNM1 gene caused the disease in a clinic patient, Rangasamy’s team inhibited the gene’s expression in zebrafish. Under the microscope, the affected fish embryos showed seizures within 72 hours.

“We can use this model to screen for drugs — some drugs that can inhibit the symptoms. We can use it as a therapy to treat those disorders in the children,” said Rangasamy.

Although these are early days, Rangasamy said he hopes to screen for drugs soon — possibly as early as March.

Aside from zebrafish, researchers rely on an array of model species, often in sequence, from cell lines through human trials. Sometimes, as Rangasamy explained, they overlap.

“These complementary models help us get to specific drugs, because when you see in two systems that the drug works, you might be pretty sure that, OK, this seems to be a lead, or an interesting compound, that can be tested," Rangasamy said.

Where zebrafish research is concerned, there are plenty of other fish in the sea, including labs at Arizona’s three major universities. An Arizona State University researcher used zebrafish to test immune response to nanomaterials. Northern Arizona University used them to study arsenic exposure’s effects on breast cancer treatment and survival. The University of Arizona Cancer Center used them to research potential treatments for ototoxicity — chemotherapy-related hearing loss.

“Every university, if they have good biology departments, they usually have fish facilities — not just zebrafish, some other fish types as well,” said Han.

For research associates in genetics labs, that means adding another item to their task lists, somewhere between extracting DNA and performing diagnostic tests: “feed the fish.”