Rise of the Mutant Zebrafish

In the back of Peter Lyons’ office sits a tank of zebrafish. Tiny black-striped fish from the pet store. Zebrafish are popular pets, especially the ones that glow, but these aren’t family pets. They’re model organisms for genetic research that could eventually help cure diseases like epilepsy, and they’re a vital part of the research Lyons is initiating at Andrews.

Lyons has been working on a set of particular enzymes, the carboxypeptidases, for the past few years. He first started working on the enzymes while completing his doctorate at Dalhousie University in Nova Scotia, and continued the research at Albert Einstein College of Medicine in New York. He then brought his research with him, in the form of preserved cell lines he brought in liquid nitrogen from Einstein.

A Brief History of Enzymes

Enzymes catalyze the millions of chemical reactions that propel the body, and carboxypeptidases are a type of enzyme that chop off little pieces from proteins or break down old proteins. Carboxypeptidases have a very specific function—they only clip amino acids at the end of a protein chain, and they’re often found at very specific locations in the body. The carboxypeptidase that scientists know most about is a pancreatic enzyme, mostly involved in digesting your dinner. Although many carboxypeptidases’ molecular function is known, their larger role in the body is still a mystery.

Carboxypeptidase-A6, for example, appears to be linked to epilepsy. Researchers have observed a group of families in Morocco that have both temporal-lobe epilepsy and a number of genetic mutations. “We’re quite sure that it’s the mutation of CPA6 that’s causing epilepsy because only the affected family members have the mutation,” he says. “It’s not entirely clear what the regular gene is doing to prevent epilepsy normally—there’s a possibility that it could be involved in modifying neuropeptides, but we haven’t worked out which ones and where.” Carboxypeptidase- O is even more elusive: it seems to have a function in the digestive system, but Lyons and other researchers are still in the process of narrowing its function down.

For these highly specialized enzymes, these gaps in knowledge are what keep researchers up at night. “The big question for many of these enzymes are what are their substrates, where do they function, what happens if they’re messed up, and what’s the result in an organism?” says Lyons. That’s where the zebrafish step in (or swim in) and serve as model organisms to study the function of these enzymes.

Behold the Zebrafish

The tiny zebrafish are remarkable organisms. They’re small and easy to raise, they produce lots of embryos on a weekly basis, and these embryos contain organ systems that are fully functional in a matter of days. Since zebrafish are vertebrates, scientists often use them as model organisms when exploring genetic function and expression. “They have many of the same organs as we do,” says Lyons, “and scientists use the zebrafish model system to figure out all aspects of health and development.”

Zebrafish and humans have about 20,000 genes in their genomes, and scientists have sequenced both genomes. We share many of the same genes with the little fish—including the 25 or so types of carboxypeptidases. Because it’s neither practical nor ethical to do exploratory genetic research on humans, zebrafish provide the perfect model organisms to understand more about what carboxypeptidases do.

Much genetic research involves isolating a gene, modifying or mutating it in some way, and returning it to an organism to observe how the changed gene is expressed. The expressed mutations help determine what the consequences might be if the gene naturally mutates, but may be analyzed within the controlled context of a model organism. In general, “enzyme functions in the fish might indicate what it does in the human,” says Lyons. So if Carboxypeptidase-O shows up in a zebrafish’s intestine, researchers know where to begin looking for that same enzyme in humans.

In the Lab at Andrews

Lyons is in the process of setting up Andrews’ labs to do this kind of genetic work on zebrafish. Zebrafish are quite finicky, and require well-controlled temperature, light and clean water, in addition to a defined night and day cycle. So the zebrafish research is on hold while he sets up the proper equipment. The tank in the back is just to see if his zebrafish can produce the quantity of embryos he will need—and they’re also fun to watch.

Once he gets the zebrafish installed in their research tanks, he’ll begin collecting their embryos. Under a dissecting microscope, he inserts a gene encoding a modified carboxypeptidase enzyme into the embryo with a fine-tipped glass capillary to create what will become a transgenic zebrafish. Transgenic zebrafish, modified to express a fluorescent gene, are sold as “glofish” in pet stores. Lyons is doing something similar, but his fish express effects of modified carboxypeptidases rather than fluorescence.

At the moment, he’s having some problems with his fish. Every morning, he puts them into a tank with a slotted bottom to separate out their eggs. Zebrafish will eat their eggs if they aren’t removed from the tank just after birth, and Lyons’ fish are managing to get through the slots and eat all the embryos. “I need a finer filter,” he says ruefully, looking at the complacent little cannibals.

Once the lab is up and running, Lyons can begin investigating physical expressions of mutated carboxypeptidase. In the meantime, he’s doing his experiments on his cell lines. A cell line is a group of cells that “have been immortalized—in theory,” says Lyons. His cells, all genetically identical, can be stored indefinitely and reproduced in petri dishes. For some kinds of genetic research, “it’s easier to look at a single cell type in a dish rather than a whole organism,” he says.

The open-endedness of his research has always excited Lyons. “There’s no end to the ideas. I have seemingly limitless ideas of things we could do with the cell-culture system, and transgenic zebrafish available on the market, but there is sadly an end to the money and hands to pursue it,” he says. Thankfully, Andrews is able to help with some of that: Lyons has been assisted by several biology students over the past year, and is optimistic about Andrews’ lab capabilities. “The biology department is set up really well for this kind of research,” he says. “We can pretty easily support the research I do—not all of it requires fancy equipment, and the high-tech stuff is often outsourced to larger laboratories.”

Lyons’ research has also earned him funding from several sources, including the Natural Sciences and Engineering Research Council of Canada, the National Institute of Health, and the neuroscience nonprofit The Grass Foundation.

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