Sharks, salt (and a taste of lobster)

An intensive week at the bench in Maine introduces students to modern lab strategies and techniques, ancient DNA and a clambake to write home about.

Few medical students can say that, as part of their education, they plucked a writhing dogfish shark from a pool of water. Or that they ended their first year by seeking clues to human disease in the organs of a fish that reached its evolutionary peak more than 300 million years ago.

This past June that pool of sharks lured about two dozen
Yale students to Salisbury Cove, Maine, just a few miles from Bar Harbor. For more than a century, scientists at the Mt. Desert Island Biological Laboratory here have explored the genes shared by fish and humans. For about half its lifetime, the laboratory has had strong ties to the School of Medicine, a relationship that began in the 1950s, when Franklin Epstein, M.D. ’47, HS ’49, professor of medicine, began bringing residents and fellows to Maine. In June 2000, for the first time, medical students arrived for a week’s training in bench research. This year another group of students repeated the Intensive Pedagogical Experience, a program designed to introduce them to the strategies and techniques of laboratory science and to encourage them in careers as physician-scientists. Most of the students arriving in June had just completed their first year of medical school; the group also included a student who began med school this fall.

In the course of their week on Mt. Desert Island, the students cloned genes, learned to synthesize DNA by means of polymerase chain reaction and generated copies of proteins by inserting RNA into frog eggs. They also learned the difference between Western blots, used to find specific proteins, and Southern and Northern blots, used to find the RNA and DNA for specific genes.

Rupali Gandhi, who is beginning her second year at Yale this fall, majored in biology as an undergraduate, then went on to get a law degree at Yale before taking up medicine. “I hope to come away from this course with a better understanding of a lot of research methods so that the next time I read a journal article my eyes won’t glaze over the methods section,” she said, sitting on a porch outside one of the many laboratory buildings scattered throughout the forest. Understanding research strategies and gaining familiarity with the scientific method are among the goals of the course, according to its director.

“The idea of rigorous pursuit of a question and a clear hypothesis is applicable to all that students do in research at Yale,” said John N. Forrest, M.D., HS ’67, who heads the Office of Student Research at the medical school and is director of the Mt. Desert Island Biological Laboratory, where he has spent the last 32 summers.

Long before the first group of Yale students traveled to Maine, the notion of an intensive laboratory experience was brewing
in the mind of Dean David A. Kessler, M.D. As an undergraduate at Amherst College, he spent four weeks one summer at the Woods Hole Marine Biological Laboratory. The time he spent studying macrophage inhibition factors in sea urchins served as a model for the program in Maine. Kessler wanted to provide students with both hands-on laboratory experience and a chance to interact closely with faculty. “You get to see things you just don’t see sitting in a lecture hall,” he said. “This is what education should be.” And holding the course on Mt. Desert Island freed students and faculty from the day-to-day distractions of being on campus.

Forrest scheduled the course for early June, after students have completed their medical school course work and before the lab’s high season in July and August, when its year-round population of 17 staff members swells to more than 200 principal investigators, postdocs, graduate students and laboratory technicians.

Amidst the laboratory’s 350 acres of poplar, pine, spruce and cedar forest, the hierarchy that typically rules academia takes a sabbatical. First of all, the laboratory, with its shingled and weathered buildings in the woods, looks more like a fishing camp than a research center for top scientists from more than 50 institutions. But enter those buildings and you’ll see they’re filled not with lobster pots and fishnets, but with beakers, pipettes, computers, centrifuges and a gene sequencer. Dress is informal. As often as not, students and faculty wear T-shirts, shorts and sandals. They spend most of their waking hours together. And, most troubling for some students, only first names are used. “We all know him as Dr. Forrest because he’s the head of research,” said student Dena Springer. “We come here and he’s John.”

At the heart of the students’ experiments is the rectal gland, an organ unique to sharks and other fish. Local fishermen provide a regular supply of spiny dogfish sharks, also known as Squalus acanthias, a relatively benign member of the shark order. Between 16 and 20 inches long, they are decidedly passive compared to their great-white cousins. Students say these fish will bite, but only if you stick a finger in their mouths. Their rectal gland is a highly specialized organ whose only function is to pump salt. Among the gland’s virtues is its relatively large size, about an inch long, making it easier to manipulate for neophyte researchers than cells or molecules.

Examining a fish’s ability to control its salt may seem a highly esoteric field of study. Yet the rectal gland offers clues to understanding cystic fibrosis, the most common fatal childhood disease and one that is also concerned with salt imbalance. It is caused by a genetic disorder, the malfunction of a protein called the CFTR chloride channel, which renders the body unable to regulate chloride transport. Without the proper functioning of this protein, a sticky mucous secretion develops, clogging lungs, sinuses and the digestive system.

Working in four groups of six, the students approach the gland from various perspectives. Over the course of a week they try to answer these and other questions: What is the function of the rectal gland in the shark? How is secretion by the gland regulated by known agents and second messengers? What is the function of CFTR? Can we determine Na-K-Cl cotransporter activation in an isolated cell preparation? What is the importance of single nucleotide polymorphisms in the post-genome era?

Students may look at the entire organ, measuring its function with agents such as barium, forskolin or IBMX, which inhibit or stimulate chloride transport. For another experiment, working with tissue from the gland, they isolate a protein and try to turn on and off its capacity to transport sodium, potassium and chloride. They may phosphorylate the protein and look for signs of activity. When they look through a confocal microscope, the students expect to see a lot of green, a sign that phosphorylation has activated the cotransporter protein. They also take the CFTR chloride channels they’ve generated in frog eggs and measure their electrical activity. Continuing that experiment, they add hormones to open and close the channels.

The science behind the experiments was complex, and at times the mechanics could be demanding and repetitive. One rainy afternoon medical student Jenny Blair watched as a shark’s salt gland excreted liquid into a narrow-bore pipette several inches long. When the pipette filled, every few minutes or every few seconds, depending on whether chemical agents were involved, she inserted a new pipette into tubing coming from the gland, while others on her team measured the amount of liquid, then determined how much chloride it contained. Upon completing the experiment, they calculated the gland’s ability to excrete chloride under the conditions studied.

Part of the students’ fascination comes from knowing that another team in another lab or rotation will pick up on their experiments. “We took the body parts of a shark and extracted RNA from them,” said student Benjamin Negin, describing a typical research sequence. “Then we passed the baton on to the next group. They’re turning the RNA into DNA. The final group is using polymerase chain reaction of the DNA to identify what it is.”

Niya Jones and Bao Duong were part of the second team in the sequence Negin described. They learned how to manipulate a pipette to insert shark DNA into an agarose gel. They were filtering the DNA by size, looking for the genes related to a family of proteins that regulate chloride transport. Steve Aller, a doctoral candidate at Yale, guided them through the process, teaching them how to hold the pipette so the DNA slips into a well in the gel without smearing.

When, at the end of their experiment, they found a set of small genes they could not identify, Jones and Duong passed them off for further research to the next team in the rotation. “If it’s a subunit of the CFTR channel or an unrelated protein, it would be interesting to see,” said Spencer Epps, who planned to compare the genes to those in an online database at the National Institutes of Health.

Not all the experiments yield clear results. In presenting their findings students were refreshingly honest about the successes and failures of their experiments, as well as the unanswered questions. “We don’t have wild-type data,” said one student whose team explored mutations in ion channels, “because it got messed up.” “The sequencing did not work,” said a member of Epps’s group, which had hoped to identify unknown genes, “and we’re not sure why.” Most experiments proved successful, insofar as the students noted patterns of behavior in the way cells responded to stimulants and inhibitors. For example, John Koethe explained the slopes and valleys on a graph that tracked the rectal gland’s response to different agents. “They show barium inhibition, recovery, then inhibition,” he said. Another group working with rectal gland slices and tubules found that forskolin and IBMX activated the chloride channel, leading to chloride excretion. Working with the whole gland, one team reported inconclusive results of their experiment; a high dose of potassium inhibited chloride secretion but a low dose yielded mixed results that offered no firm conclusions as to its effect.

The experiments conducted here are a natural outgrowth of the laboratory’s focus on the physiology of marine and human organisms. Sharks, for example, reached evolutionary perfection between 300 million and 400 million years ago and have changed little since. Their genome is about 70 percent identical to that of humans. “Humans adopted many of the successful systems they evolved,” said James L. Boyer, M.D., HS ’67, Ensign Professor of Medicine and director of the Yale Liver Center, who heads the Mt. Desert Island Biological Laboratory’s board. “That is why we are able to use those marine creatures for relevant research on human systems. The differences are far fewer than the similarities.”

Since its founding as a summer school for Tufts University students in 1898 in South Harpswell, Maine, the biological laboratory’s focus has shifted between education and research. It first taught marine biology to undergraduates, but quickly became a center for marine research as well. In 1921 a land-holding organization, Wild Gardens of Acadia, offered the lab 100 acres on Mt. Desert Island. George B. Dorr, one of Wild Garden’s leaders, was instrumental in founding Acadia National Park and had a vision for Mt. Desert Island. “He believed this pristine setting would be an ideal circumstance for the study of nature,” said Jerilyn Bowers, director of development and public affairs at Mt. Desert Island Biological Laboratory. “He wanted this to be a very cultural, artistic society.”

The laboratory was founded at a time when scientists had embraced Darwinian thought and looked for answers to human biology in the sea, where life began. Dozens of marine biology labs sprang up, but only three in New England have lasted: Woods Hole, Cold Spring Harbor Laboratory in New York and the Mt. Desert Island Biological Laboratory. Of the three, only the Mt. Desert Island laboratory continues to study links between fish and humans. Among its neighbors on Mt. Desert Island is The Jackson Laboratory, the world’s largest mammalian genetic research facility, which specializes in breeding genetically engineered mice for research. As a research center the Mt. Desert Island Biological Laboratory has brought forth significant discoveries, said Bowers. “Much of what we know today about how the kidney functions came from early research conducted here,” she said. The CDNA of several sodium chloride transporters was first cloned here by two scientists now at Yale, Steven C. Hebert, M.D., chair of the Department of Cellular and Molecular Physiology, and Biff Forbush, Ph.D., professor of cellular and molecular physiology.

A quarter-century ago the laboratory renewed its educational focus by offering programs for both graduate and high school students, along with conferences and symposia for scientists from around the world. Still going strong is an eight- to 10-week summer science program for high school students from surrounding Hancock County. (One of those alumni, Aller, is now a Ph.D. candidate in molecular biophysics and biochemistry at Yale and an instructor in the summer program for Yale medical students.) It is also strengthening its research component with a five-year plan to recruit three scientists who will be the laboratory’s first year-round researchers.

Throughout the years, according to those who know it well, the lab worked its magic through an informal environment that brought together biologists, physicians, basic scientists and students and, by its remoteness, encouraged cross-fertilization of ideas. “It is more conducive to learning and picking up different approaches than if you just stay with your own groups,” said Boyer. “At Yale,” he added, “you have to go out of your way to make those interactions. Here, you can’t help but do that.”

For medical students, it is these interactions that make the experience worthwhile. In their evaluations of the program’s first year, students said that the opportunity to work in small groups with faculty was one of its most attractive features. Rupali Gandhi, the student now starting her second year of medical school, agreed. “You can ask as many questions as you want as many times as you want,” she said, “and get very
clear explanations.”

Joining Forrest on the faculty this year were Forbush and Raymond Frizzell, Ph.D., chair of the Department of Cell Biology and Physiology at the University of Pittsburgh. (Frizzell arranged a similar program this year for medical students from Pittsburgh, who arrived a week before the Yale students.)

If the first year of the Intensive Pedagogical Experience offered any lessons, it was that the schedule left students little time to enjoy Mt. Desert Island. This year, students had a free afternoon and evening every other day. During those free hours Forrest and Forbush led bicycle and hiking tours of the island.

On Friday, the last day of the course, students made their final presentations after lunch and had the rest of the day to themselves. That evening Forrest organized a clambake at Seawall, a picnic area and campground at Acadia National Park in a section of the island the locals call the “quiet side.” Forrest arrived early to stoke a bonfire for cooking. Before long, lobsters and bags of vegetables and mussels were steaming in huge pots of seawater as the sun set over the rocky shore.

Earlier in the week Forrest sat on a wooden chair outside his lab on a bluff overlooking Mt. Desert Island’s Eastern Bay and mused about the course. One of its virtues, he said, is a “conference room” like this, in the shade of a tree with the ocean below. “It’s asking a lot,” he said, “to say you’re going to change people’s lives in a week, that people are to change their career goals based on a week’s experience. But this course has the potential to encourage people who otherwise might not think about a research career. Some of these students have never run a gel, ground up tissue or looked at cells under a microscope in a research mode before. Here we have very bright students learning all this medicine from lectures, books, videos and the Internet. Unless they are looking inside a cell, thinking about how to study some component of how that cell functions, unless they are striving to interpret what really exists, from their own questions, they are going to be passive recipients. The idea of hands-on work is often so illuminating that it leads people to say, ‘This is what I want to do for the rest of my life.’” YM

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John Forrest

Professor of Medicine

James Boyer

Ensign Professor of Medicine (Digestive Diseases)