Lessons from the depths

Accounts of death row inmates released from prison based on DNA evidence have become as routine as news stories about celebrity births, betrothals and breakups. On television, the popularity of shows such as CSI, Crossing Jordan and Cold Case Files attests to the public’s familiarity with this field of research.

In 1983 Kary B. Mullis, Ph.D., who 10 years later was a co-winner of a Nobel Prize in chemistry for his discovery, enabled this revolutionary application of genetic science with a method called polymerase chain reaction (PCR), which allows scientists to analyze DNA samples by generating copies of a genetic fragment. But the process, which relies on the enzyme polymerase, was as time-consuming as it was groundbreaking. DNA strands had to be unwound and separated through a three-step process involving about 40 heating and cooling cycles. It wasn’t until scientists figured out how to streamline the technique, through a method called rapid automated PCR, that it became the medical and forensic bonanza it is today. What made the breakthrough possible was the identification of a bacterium, Thermus acquaticus, which thrives in hot springs. Its polymerase can survive temperatures that fluctuate between 72 and 90 degrees Celsius.

This is just one example of how scientists have expanded research capabilities by shifting their gaze from earth to water.

The guinea pig was once synonymous with biological research. Rats and mice are still ubiquitous in research labs, but researchers are increasingly pulling up anchor to find aquatic animal models that might offer better solutions to specific research questions. At Yale several scientists are embracing their potential, using aquatic specimens to study everything from liver and kidney diseases to neurophysiology and the effects of toxic substances on living organisms.

James L. Boyer, M.D., HS ’67, FW ’69, director of Yale’s Liver Center, has spent the last 33 summers conducting research at the Mount Desert Island Biological Laboratory in Salsbury Cove, Maine. Recently Boyer has been studying the skate liver to see how it sheds itself of toxic substances. He initially used the livers of dogfish sharks, but found the skate liver easier to handle and more mammalian in size and shape.

Boyer, who is the director of the Comparative Toxicogenomics Database at the Mount Desert Island facility, says scientists long believed that if you wanted to learn about human biology, you had to study other mammals. Now researchers are finding that studying less-similar creatures can also be illuminating. “By comparing our genes with the genes of lower vertebrates we can often get a better estimate of what the most important parts of our genes are, because the differences are greater than when we compare our genes with genes of a mouse or other mammal, which are very similar if not nearly identical,” he says.

According to Boyer, the most famous example of an aquatic specimen shedding light on human physiology is the squid axon, which is so large that when scientists probe it with electrodes they can easily see how nerves conduct signals. Another is the sea slug. With its large cells, it has helped scientists understand signaling pathways in nerve tissues.

At the Mount Desert Island lab, scientists have spent years studying the rectal gland of the dogfish shark. This gland has one job only—to pump salt. Their research has provided insights into such diseases as cystic fibrosis, which stems from a salt imbalance caused by the body’s inability to regulate chloride transport. “There are many examples of this type of story,” Boyer says, “but it’s not yet appreciated how useful these species can be.” Aquatic animal research has lagged, he says, partly because the study of marine specimens requires travel to remote locations as well as special facilities and equipment.

The search for clues to human physiology in the sea dates to the early 20th century, when scientists, inspired by Darwin, founded marine biology labs, including the Mount Desert Island facility and the Woods Hole Oceanographic Institution in Massachusetts, along the East Coast. But, according to Boyer, it wasn’t until the genetic revolution—the cloning and sequencing of genes—that the use of marine specimens for research really gained currency.

“Through comparisons of genes from lower organisms with human genes, we demonstrated that our genetic material was more similar to these lower vertebrates than we thought,” Boyer says. The gene that codes for the bile salt export pump in the skate liver, for example, is 70 percent identical at the amino acid level to the human gene. “We find that all of the human mutations occur in the same regions that are identical between skate and man,” Boyer says. “Thus, we are beginning to learn that the 30 percent of our bile salt export gene that is different from the skate is not very important from a functional point of view.”

Scientists now have a pretty good library of mammalian cell lines, but according to Boyer, similar models need to be developed in aquatic animals. That is a priority for researchers at the Mount Desert Island lab, who have a grant from the National Institute of Environmental Health Sciences to create the Comparative Toxicogenomics Database.

A new model from India

The current poster fish for aquatic animal research is the zebrafish, a small freshwater fish originally found in slow streams and rice paddies in the Ganges River in India. In the early 1970s, George Streisinger, Ph.D., at the University of Oregon, determined that zebrafish were an invaluable model for studying vertebrate development and genetics. Since then, their embryos have been used worldwide to study how all vertebrates, including humans, develop.

What makes the zebrafish ideal is their eggs—they are transparent and they develop outside the mother’s body. And while a mouse takes 21 days to develop, zebrafish grow from a single cell into a tiny fish within 24 hours. Scientists can watch under the microscope as the zebrafish cells divide and form different parts of the infant fish’s body. Scientists can easily move or destroy a cell to see what happens. And zebrafish, like humans, have a backbone, making them more similar to humans than commonly studied invertebrates such as Drosophila and C. elegans.

Zebrafish are also easy and relatively inexpensive to maintain, manipulate and monitor in the lab. This makes large-scale studies far more feasible and affordable. They thrive in many environments, can be kept together in large numbers, are easy to breed and require less stringent research protocols than mammals. In fact, the zebrafish has become so popular in recent years that it has its own magazine, Zebrafish, and, like all celebrities, its own website, www.zfin.org.

Zhaoxia Sun, Ph.D. ’98, established the first zebrafish facility at the School of Medicine in 2003 in a former autoclave dishwashing room down the hall from her lab. It now resembles an aquarium, with shelf upon shelf of shoebox-sized tanks, each swarming with tiny striped fish. Sun, an assistant professor of genetics, returned to New Haven in 2003 from a postdoctoral fellowship at the Massachusetts Institute of Technology, where she used zebrafish to explore the genetic causes of polycystic kidney disease.

“Zebrafish provide so many unique features,” Sun says. “So many things not previously possible are now possible.” In collaboration with researchers at MIT, she performed a large-scale zebrafish screen and identified 12 genes that, when defective, can cause polycystic kidney disease. “It would have taken a lot longer without the zebrafish,” Sun says.

Her zebrafish studies are starting to attract the attention of other Yale researchers, and she has received inquiries from faculty in nephrology, physiology, genetics and cardiology about possible collaborations. She welcomes these opportunities. “I think zebrafish will be helpful in bridging different fields,” she says. “It’s still a young field, but it’s a field with huge potential.”

Across campus, Scott A. Holley, Ph.D., assistant professor of molecular, cellular and developmental biology, is the only other Yale scientist working exclusively with zebrafish. Holley, who studies early vertebrate development and genes related to skeletal defects, meets regularly with Sun, and the two plan to teach a course together. “The field is in a rapid growth phase,” says Holley, noting that at his first zebrafish conference in 1997 about 120 scientists attended. At a meeting in 2004, attendance topped 1,000.

A tool from the coral reefs

Vincent A. Pieribone, Ph.D., associate professor of cellular and molecular physiology and neurobiology, credits the green fluorescent protein (GFP) found in Aequorea victoria jellyfish with revolutionizing his studies of how the human brain works. He wanted to see how collections of nerve cells fire, but the density of cells in the mammalian brain and the speed at which they fire make it next to impossible, and recording one cell at a time “was like listening to an individual phone line in New York City and trying to figure out from that how the city functions,” he says. Plus there was the challenge of recording what was happening without resorting to invasive, damaging procedures.

Enter GFP. “You can put it into any cell in any animal and the cell will fluoresce green and be identifiable,” Pieribone says. “It’s been hugely important to scientific study.” GFP has been used to track cells in a wide range of animals and has enabled scientists to watch cells develop in real time without having to kill the specimen. Kaeda-type GFPs have taken the promise of this research tool one step further, because they work as “reporters.” “They don’t just tag cells,” Pieribone says. “They can be made to change color (from green to red), allowing scientists to monitor the movement and synthesis of the protein.” Pieribone’s laboratory is focused on fusing GFPs to other proteins that cause the fluorescence emission of GFP to be altered by a cellular process. “This tells us what’s going on—is the calcium going up, for instance—when the cells do what they do.”

The success of GFP from jellyfish prompted scientists to return to the sea in search of proteins in other colors. In June 2001, on Lizard Island, part of the Great Barrier Reef in Australia, Pieribone and his colleagues identified two corals (Lobophyllia hemprichii and Flavites spp.) that produce fluorescent proteins. One glows red; the other switches from green to red when exposed to UV light. “A lot of biological specimens have backgrounds that fluoresce a kind of greenish glow,” Pieribone says, “so with these new fluorescent proteins, things stand out better. They’re a lot easier to see.”

In all, he has identified about 40 species of fluorescent coral. The two GFPs Pieribone and his colleagues cloned are not currently in use, but others like it are. Besides corals and jellyfish, scientists have also cloned GFP-like sequences from anemones, sea pansies, sea pens and copapods, bringing the total number to around 70.

Along with new fluorescent proteins, Pieribone also found that coral reefs the world over are dying, which he says would be a huge loss for science, to say nothing of the ecological ramifications. “When we lose these reefs, we’ve lost an amazing library of genes,” Pieribone says. To save what he and other scientists consider a vital resource, Pieribone is trying to apply biomedical technology to understand how global warming is killing the corals. While this is a diversion from his work as a neurophysiologist, he says there’s an important connection. “Nature makes mutations that can be cloned and studied for utility in the laboratory,” he says. “If we kill the animals, we lose that ancient library and we don’t have millions of years to wait around for another to be formed.”

Pieribone’s work with aquatic specimens prompted him in September 2003 to found Marinus Pharmaceuticals, based in Branford, Conn., which has seven employees. The company seeks to use information gathered through marine research to create drugs to treat epilepsy, depression, schizophrenia and heart disease. “The environment in the ocean is harsh. Organisms that have adapted to survive there are very tough,” he says. “They have a lot to teach us about survival strategies.”

Sea hares and nerves of steel

As a researcher who studies nerve cell function, Leonard K. Kaczmarek, Ph.D., professor of pharmacology and cellular and molecular physiology, is particularly fond of the squid and the sea hare. Their main appeal is their large cells. Some of the sea hare’s nerve cells, for example, can be seen with the naked eye. “They’re also color-coded, from white to bright orange,” Kaczmarek says, “so you can give each one a name and number. It’s very easy to figure out how nerves control behaviors.” Sea hares are also desirable research specimens because their cells are so hardy. “The human brain, if deprived of oxygen, dies in a few minutes,” Kaczmarek says. “Sea hare nerve cells have been known to live up to a week.” Using this species, Kaczmarek is studying bag cell neurons, which serve as a master switch for the control of reproductive behaviors.

Despite the differences between humans and marine organisms, the way nerve cells respond to the outside world and generate electrical activity that controls behavior is highly conserved across evolution. For example, many ion channels—the proteins that control the electrical behavior of neurons—are almost identical in molluscs and humans.

The main attraction of the squid, Kaczmarek says, is that its synapses are so large they can be seen without a microscope. Many human synapses are round and measure 1 to 2 microns in diameter. The squid giant synapse is 300 microns wide and as long as a millimeter. “It makes it very easy to study how one neuron can stimulate another neuron. It also allows us to investigate ion channels in different parts of the synapse in ways that are simply not possible with mammalian cells.”

Although he uses rats and mice for most of his research, Kaczmarek sees himself continuing to study sea animals for the foreseeable future. “So many breakthroughs happen with simple model systems,” he says. “Besides, it’s very nice to have both perspectives.”

Like Boyer and Pieribone, Kaczmarek worries that funding, fashion or affronts to the environment could threaten marine animal research. This, he says, would be a shame. Noting that research based on squid led to Nobel prizes in 1963 and 1970, he says, “Just look at the history. It’s very obvious that looking at really simple systems gives insights into understanding more complicated systems.” YM