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12/12/96

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Background: The science behind the gene for sexual behavior

The discovery of a gene that controls most or all sexual behavior in male fruit flies comes out of a collaboration among scientists at four universities. Each lab has a different expertise in molecular genetics, neurobiology and animal behavior. The consortium was formed in 1993 after an international scientific meeting, where the scientists found that they had all been studying the same gene and that it might offer a key to understanding the links between genes, brains and sexual behavior.

In the Dec. 13 issue of the journal Cell, the four laboratories publish the results of a joint study that shows that the gene, named "fruitless" and nicknamed fru, works in a small number of brain cells to command all, or nearly all, aspects of the sexual behavior of the male fruit fly. The scientists say that to the best of their knowledge, this is the first time anyone has identified a single gene that clearly acts in its normal role to specify all, or nearly all, aspects of a complex behavior in an adult animal.

The four laboratories are led by Bruce Baker, professor of biological sciences at Stanford University; Steven Wasserman, associate professor of molecular biology and oncology at the University of Texas Southwestern Medical Center in Dallas; Jeffrey Hall, professor of biology at Brandeis University; and Barbara Taylor, assistant professor of biology at Oregon State University.

The fru gene was cloned in a cooperative effort between the Wasserman and Baker labs. Wasserman's group discovered that the piece of DNA that codes for the fru gene is unusually large. Using the tricks of molecular biology and genetics, the two labs pieced together a "road map" of this long string of DNA.

Baker's lab discovered the products of the gene, and found that it makes transcripts, or messages, that appear to control the actions of other genes. In collaboration with Hall's lab, they showed how fru operates as part of a group of genes that work together to control all aspects of sex in the flies.

Hall's laboratory at Brandeis University, in cooperation with Baker's group, documented the behavior of flies with many different laboratory-induced mutations in the fru gene. They showed that the normal functioning of the gene is necessary for nearly all aspects of male sexual behavior.

Taylor's laboratory at Oregon State University found that the fru gene functions only in the central nervous system. It controls sexual behavior by acting in only about 500 of the fly's 100,000 nerve cells. Based on the location of these cells, the scientists say the gene may operate by programming the neurons that command and control other nerve cells and muscle cells during sex.

The scientists say that fru provides them with a tool to learn a great deal about how genes work in the nervous system to specify sexual behavior.

"Showing that a behavior as complex as sexual behavior is controlled by a single gene, at least in flies, raises the obvious possibility that other behaviors will be similarly controlled," Baker said. "Studying such genes may give us insight into the functional organization of the central nervous system."

So far, a comparable gene to fru has not been found in other species, including humans. The researchers said that they expect scientists to look for such genes ­ and if they are found, to look at whether and how the genes influence behavior.

It also is not known whether fru influences the behavior of female flies. Sex-specific transcripts of the gene are expressed in most of the same areas of the brain for females as for males, but female sexual behavior in Drosophila is more subtle than in the male and has not been well studied so far.

Now that fru has been cloned, it should have practical uses as a scientific tool. It may also prove valuable in agriculture, where some other species of fruit flies destroy crops. It could give pest control experts a reliable way to raise large numbers of sterile insects, as "birth control" to prevent the spread of an infestation of flies. Stanford and UT Southwestern have applied for a patent on the gene.

Following are additional details about the discovery:

How fruit flies behave

The fruit flies named Drosophila melanogaster are the "laboratory rats" of the insect world. They are the animal most well studied by geneticists, in part because they reproduce and grow rapidly, so that scientists can mutate a gene and quickly see what happens to flies that lack that particular gene. Among the gains of 80 years of Drosophila genetics are insights about how the body plan of a fly is laid down as the animal develops from a single cell to an adult. These same processes turn out to underlie the development of other animals, including humans.

Scientists also have studied Drosophila's behavior. Hall has led in the development of techniques to document how flies normally act during courtship and mating. His lab and others have shown that from the moment a male fly senses an eligible female by sight and smell, he courts her with a series of gestures as patterned as a dance ­ following, stroking, licking, and sticking out a single wing to play a rhythmic courtship song before he bends his abdomen and attempts to mate.

In the early 1960s, Kulbir Gill, an Indian scientist then doing research at Yale, noticed that when fruit flies were treated with radiation to cause genetic mutations, one strain changed its courtship pattern. Male flies with a particular mutation are unable to distinguish females from males as appropriate partners for courtship. The males of this type court both sexes, but are unable to carry courtship through to mating. Sometimes these males form long chains, with each male courting the fly in front and being courted by the fly behind it.

The mutation was indirect evidence that there must be a normal gene for sexual orientation, or at least for the fly's ability to tell whether other flies are male or female. Because the mutant flies left no progeny, in the tradition of Drosophila genetics, the normal gene was named after its mutation ­ dubbed "fruitless" and shortened to fru.

According to Hall, this gene was mostly a curiosity until recently. However, both his lab and Taylor's lab have done several studies on it. One of Hall's discoveries was that the fru-1 mutation not only causes male "chaining" behavior, it also subtly changes the fly's courtship song. He also discovered a second mutation of the same gene. When other geneticists came across fru in their work, they often discussed it with him.

That is how both Baker and Wasserman happened to contact Hall.

A family of genes for sex

Over the past 15 years, Baker's lab at Stanford and a handful of other labs have revealed the mechanisms that control sexual differentiation in fruit flies. This work has shown that a single gene named "doublesex" receives instructions from two other genes, which are in turn controlled by a master gene at the top of the hierarchy. Depending on the instructions that doublesex receives while a fly is an embryo, it will develop the sex organs and other sexual characteristics of a male fly, or of a female. The data were consistent with the idea that the doublesex gene controlled all aspects of sexual differentiation.

However, in 1992, Taylor conducted studies on a muscle that appears only in males and develops only if it is properly connected to a certain neuron. She found that this muscle's development is not controlled by doublesex, though it is governed by the genes higher up in the sex differentiation hierarchy. Taylor pointed out that this meant there had to be an as-yet undiscovered gene that controlled this aspect of sex.

"Barbara's work established very clearly that there must be another gene in the hierarchy," Baker said. "At that time, we had no idea that the missing gene would be anywhere near as interesting as it turned out to be. We had no inkling that it would control male sexual behavior."

In Baker's lab, research associate Lisa Ryner had a hunch about how to find the missing gene in the sex hierarchy. She reasoned that it might contain patterns in its genetic code similar to doublesex, and designed probes to find other genes with those patterns. In 1993, Ryner's probes found the right gene.

She also discovered, from its location on the fly's chromosomes, that the mystery gene was likely to be fru.

A long chromosomal walk

During this same period, Wasserman's lab had been working with others, carrying out a comprehensive search for mutations that make fruit flies sterile. Wasserman specializes in the genetics of male fertility, and he was looking in particular for mutations that interfered with the formation of sperm. But the search also found two new mutations that caused the same "chaining" behavior observed among fru male flies.

The flies had normal anatomy and sperm, but they were behaviorally sterile ­ unsuccessful at passing on their genes. While they courted females as well as males, they did not attempt to mate in either case. These flies turned out to carry new mutations of the fru gene.

"Because of the way these new mutations were made, we realized that we had new molecular markers that would allow us to clone this gene," Wasserman said. Talking with Hall and Taylor, he also realized that evidence was mounting that fru might do its work by acting in the adult nervous system ­ a part of the fly that had been relatively little studied by geneticists. He encouraged M.D./Ph.D. student Diego Castrillon to do a "chromosomal walk" ­ a step-by-step assembly of the ladder of the gene's DNA ­ and to map the sites of the mutations in the gene.

That turned out to be a daunting task. A typical Drosophila gene contains about 2,000 base pairs, or "rungs" of the DNA ladder. The fru gene has at least 70 times that many rungs ­ 140,000 base pairs.

Findings from four labs

At the annual meeting of Drosophila geneticists in the spring of 1993, the Baker and Wasserman groups quickly realized that they were cloning and studying the same gene, and that Ryner's probes plus Castrillon's analysis would make that massive job more practical. They combined their efforts and cloned the entire fru gene. (Scientists in Japan also have cloned some segments of fru, in research reported earlier this year.)

"However, the research reported in Cell goes far beyond cloning a gene," Baker said. "That is because the consortium we formed in 1993 includes four labs with complementary expertise. This is a big and complicated gene and took the skills of all four to understand how it works."

In collaboration with postdoctoral fellow Stephen F. Goodwin in Hall's lab, Ryner went on to show that the fru gene is governed by genes higher in the sex-determination hierarchy, so that it makes different products in males than it does in females. She also showed that it probably acts as a transcription factor, meaning that it may govern the actions of other genes needed for specific aspects of sexual behavior.

In addition, she isolated copies of many of the transcripts of the gene ­ pieces of RNA that convey the gene's instructions to the cell.

Armed with Ryner's and Castrillon's data about the gene and its transcripts, other members of the research consortium went on to find out more about fru. Goodwin and research associate Adriana Villella of Hall's lab worked with postdoctoral fellow Anuranjan Anand in Baker's lab to make new mutations in the fru gene that revealed a lot about what the gene normally does.

Villella used video methods developed by Hall to record and analyze the courtship and mating rituals of flies that have these new mutations. She used very sensitive microphones to record and study the courting song.

The key finding of these behavioral studies was that mutations of this single gene affect all, or nearly all, aspects of the male fly's mating behavior. The stronger the damage to the fru gene, the more aspects of courtship and mating were disrupted. With the most severe mutation, the flies were barely interested in courting at all. However, they appeared normal in other ways, able to fly and walk normally. This is an indication that the fru gene primarily controls male courtship and mating behavior.

Most of the mutations influenced the first and last steps of courtship. In the first step of the ritual, the males lose their ability to tell females from other males; they court both sexes. In the final step of mating, the males don't bend their abdomens to copulate.

Some mutations affected more parts of the ritual, including the courtship song. Although these flies could fly and flick their wings, they were unable to vibrate a wing to sing normally. Some had subtle changes in their songs that may have made them unrecognizable to females. Some could not sing a note.

Behavior and the brain

Taylor took the research in a different direction. Using RNA transcripts of the gene's sex-specific messages, she designed probes to find out where in the body each of those messages was being made ­ that is, in which cells the gene was expressed, or turned on.

She found signs that the particular transcripts of the gene that control male sexual behavior were expressed in only 500 cells, all located in the central nervous system and nowhere else in the fly. This is a tiny number ­ one-half of one percent of all the fly's central nervous system cells.

While more work will be necessary to determine what the 500 cells do, Taylor's preliminary anatomical analysis shows that some of these nerve cells are located in parts of the nervous system likely to be involved in processing information from the fly's sense organs. Others are located in cells that likely function to direct and coordinate the activities that make up sexual behavior.

Three of the four labs plan to continue with the collaboration that they have dubbed the "fruitless consortium." Under a grant from the National Institutes of Health, Baker, Taylor and Hall will study how genes cause changes in the way brain cells develop, and how that affects the behavior of the flies.

"As yet we have very little information about how to link all those steps together," Taylor said. "We think this research will provide an impetus and a source for profound understandings of how the nervous system, in any organism, is functionally organized."

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-By Janet Basu-


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