Making a Muscle

Gigi Marino
June 01, 1994

Introduce Fud genes into the fruit fly, and you get no fly: The mutated embryo dies within 10 hours. But before the unlucky Drosophila expires, an electron micrograph will show muscle lesions that look like scattered buckshot instead of the slender elegant strands of healthy muscle.

"Not many people have looked at developing muscle in a fly embryo," says Susan Abmayr, an assistant professor of molecular genetics Penn State. "We're some of the first people in the country to start identifying these kinds of genetic defects."

But muscle in a fruit fly?

microscopic view of muscle

A nuisance to the non-scientist, the common fruit fly has been the geneticist's choice for well over half a century for fairly simple reasons. In genetic study, laboratory mice are often prohibitively expensive, and humans are ethically impossible. "Flies," says Abmayr, "are ideally suited. Throw them in a gamma source, feed them chemical mutagens, nobody cares."

And whereas humans have 23 pairs of chromosomes, fruit flies have only four, allowing more intense genetic study cheaper and easier. "You can't get people to mate the way you want them to," Abmayr jokes. "Their generation time isn't two weeks." A female fruit fly, on the other hand, can lay 50 eggs per day with a generation time of 12 days, which means a million or so flies can be grown in next to no time at all. Which is exactly the modus operandi of Abmayr's lab. All the refrigerators there are chock full of glass bottles, each containing a blob of creamy agar and pupating Drosophila: a good 200 different genetic alterations. In the past decade, researchers following this scheme have mapped out volumes of Drosophila gene sequences, many believed to have human homologs—genes that are homologous or similar in structure and function to human genes. For this reason, Abmayr's work with the Fud genes, by helping to explain muscle development on a cellular level, could provide a collaborative link in the understanding of inherited muscular dystrophies in humans.

The human body contains three types of muscle—smooth, cardiac, and skeletal. Only skeletal muscle (40 percent of the body), the quintessence of shapely thighs and enviable pecs, is controlled by the brain. When working at its optimum, this muscle system produces the likes of Shaquille O'Neal and Jackie Joyner-Kersee. But when there's a problem, mainly with the protein dystrophin, those same lovely muscles shrivel, twist, and degenerate. In muscular dystrophies, all muscle is affected. Most of the victims of the more severe form, Duchenne's, die from heart failure. Those who have a milder form, Becker's, can survive to middle age. No one knows why, but the muscular dystrophy gene has been located on the X chromosome, and therefore almost always occurs in males. (Females, with two X chromosomes, can compensate if one is defective.)

"Muscular dystrophies are usually observed late in development," says Abmayr. "These genes could play a part, but it's not clear. It could be that if you completely knock out the gene you won't make any muscle. If you have a defective gene, you'll make muscle, but the muscle will slowly degenerate."

Abmayr explains that muscle is the only tissue in the body that's dictated by the fusion of cells. "They're called multi-nucleate syncitia, free-floating nuclei in a common cytoplasm. It's caused by cells fusing together, two or 25, to form a syncitia—a very important part of muscle development, because specific muscles fuse to different extents." The making of muscle is predetermined and precise. Particular numbers of cells fuse to form a particular muscle at a particular neuromuscular connection, each with its own individual identity. "You've got to make the right muscle at the right place at the right time."

Abmayr began studying the making of muscles at Harvard five years ago, when she was deciding on a research area for her postdoc. At the opposite end of the country, in Seattle, a group of researchers led by cell biologist Harold Weintraub at the Fred Hutchison Cancer Research Center had just isolated a mouse gene they believed was completely responsible for muscle development. They named the gene MyoD, for myogenic (Latin for "muscle-making") determination. When injected into non-muscle cells—brain, tissue, fat—MyoD turned them all into muscle.

Abmayr was inspired to look for a Drosophila homolog. "At that time, there weren't methods available to knock out MyoD in the mouse and see what the gene was actually doing. For that reason, I thought, hey, flies. I know something about flies. It's easy to study genetic lesions in a fly," she says. "Well, not easy, but easier than in a mouse."

After a year, Abmayr and her Harvard colleagues found a fruit fly gene that was indeed homologous to the one the Hutchison group had discovered in the mouse. When the new gene proved defective, the developing fly's muscles would be pulverized into tiny bits of protein. The Harvard team named their gene nautilus. "Someone else wanted to call it nautilus," Abmayr recalls, "because, he said, 'As in Nautilus weight equipment, it's involved in building muscle.'"

In 1987, MyoD was thought to be the "master regulatory switch": knocking it out would be like flipping all the circuit breakers for muscle development. Further study has shown that MyoD, through its homologs, imparts its mysteries of musculature in every organism known to have muscle—everything from frogs to chickens to earthworms.

Yet says Abmayr, "It turns out that maybe what it's doing is a little more complex than originally thought."

When Abmayr came to Penn State two years ago, she brought her working knowledge of nautilus and a desire to understand its function. Her scrutiny led, like many scientific experiments, to a happy accident: the discovery of two new genes, both involved in muscle development, which, when mutated, make fly muscle into tiny marbles of protein.

Among Abmayr's research assistants, the mutations are sometimes called Marbles, while the genes—which in the scientific literature are refered to by the conservative moniker Fud, for fusion-defective gene—were at first known as Delilah, since they stole away strength. In the rapidly expanding world of oddly named genes—hedgehog, wingless, achaaete-scute, Antennapedia, Tin Man (which, if removed, doesn't allow for the development of a heart)—Delilah was perhaps the first feminist sobriquet. It seemed a perfect fit for their Fud gene, until Abmayr was asked to review a paper concerning a different strength-stealing gene named, yes, Delilah.

Identifying a gene follows a Sherlock Holmes approach: You begin with a body (a mutation or defect) and work backwards. But instead of answering "whodunit?" the search for the unmutated or "wild type" gene focuses on "whereisit?" The first step in identifying a gene involves precise geography. The researcher must know exactly where on a map of the chromosome the gene resides. Once that location is determined, by means of several complicated processes a clone, or isolated copy, of the gene can be made. This cloned gene becomes a useful research tool, making it possible to grow multiple, variant copies of the gene, which can then be examined to discover how the gene operates.

Such was the angle Abmayr took when she arrived at Penn State. "We were irradiating flies and looking at their progeny, specifically for the mutations of the gene we cloned." Hundreds of thousands of fly embryos must be examined under a dissecting microscope to find a few mutations. A change in eye-color is one of the markers that aids the screener. "If you treat flies with gamma radiation," Abmayr explains, "huge chunks of DNA will be lost. The gene for eye color was very close to nautilus. If the radiation knocked out eye color, it probably took out the stuff next to it." Abmayr delegated to two undergraduates the task of screening 400,000 fruit flies, looking for orange eyes instead of normal red ones.

But the mutations of nautilus she expected weren't showing up. "It was a horrible process, very frustrating," she recalls.

"Nothing was developing the way it should have. Every now and then we were seeing these abnormal embryos floating around. We thought it was our nautilus gene, but it just didn't make sense. It would disappear, then come back again. And that's because this mutation is actually on the second chromosome." Nautilus resides on the third chromosome.

The researchers tried a different tack. "Because we were looking for mutations involved in muscle, we were staining the muscles," a process that involves incubating the embryos in an antibody that recognizes myosin, one of the major muscle proteins. The antibodies attach to the muscle and give it color that can be seen under a microscope. "As a result, we actually identified genetic mutations in at least two other genes totally unrelated to the gene we cloned," which was nautilus.

Through what Abmayr calls "some tricky genetics," she was able to isolate a heterozygote (an embryo that inherits the mutated gene from both its parents) and breed up a refrigerator full of new mutations. "Now we're trying to clone them, and our long-term hope is that we'll find a mammalian homolog."

The mutation of Fud in the fruit fly affects muscle during the embryonic stage. The cells don't ever form syncitia. They don't even form muscle fiber.

"Five years ago, everyone thought MyoD was the single gene that makes muscle. Now it's turning out that muscle development is more complex than anyone thought. Here we sit with not one, but two, independent mutations that don't make any muscle.

"We've identified something that's not only important but essential. If you knock out our genes, muscle fibers are either never formed or are grossly abnormal.

"We'd like to think that the level of this gene may in some way be regulating muscle identity."

Susan M. Abmayr, Ph.D., is assistant professor of molecular and cell biology in the Eberly College of Science, 459 North Frear, University Park, PA 16802; 814-863-8254. Mary Erickson and Barbara Bour, Ph.D. candidates in molecular and cell biology, are research assistants. This work is funded by the National Science Foundation, an American Cancer Association Junior Faculty Award, and the March of Dimes.

Gigi Marino is a freelance writer and poet.

Last Updated June 01, 1994