by Giovanna Di Sauro

We have known about gay fly males for quite a while. In fact, the fruitless, or fru, mutants were initially created using X-ray radiation in 1963, and there is a 1989 paper describing the effects of the fruitless defects in males, tracking them back to a chromosomal inversion probably caused by DNA breaks and repairs following radiation exposure in the original strain. Although the BBC suggestively calls the results of a recent study conducted on these flies a “mind-control sex swap,” this is definitely not the case: one characteristic of fruitless flies is that their sexual characteristics are perfectly normal — the males are fertile and so are the females, and their bodies are not deformed. This new study completes a series of research aimed at identifying the genetic and neural components in a model of sex-linked innate behaviour — courtship in fruit flies — and has some implications for the way we think of the relationship between genes and behaviour.

What is the reason for studying fruitless flies? In Drosophila melanogaster, the common fruit fly, sexual orientation and courtship are very straightforward. The male is the only one that can be said to display courtship behaviour: when a male intends to court a female, it uses wing vibrations to produce a “courtship song.” This song can be identified because of the specific frequencies of the sounds emitted by the male. There are two “modes” of emission, known as the sine song, with a frequency of approximately 140–170 Hz, and the pulse song, constituted of brief and repetitive amplitude modulations in a range of approximately 150–300 Hz. Females respond by allowing the male to copulate (or not; whether the male is successful depends on a variety of other factors, including winning “fights” with other males).

It was already known that fruitless encodes a gene product necessary for the determination of sex-specific courtship behaviour in flies, and that the transcripts of this gene exist in two main versions produced by alternative splicing: a male version and a female version. A previous study done showed that the male version is necessary for the determination of male courtship behaviour and sexual orientation. The gene is, in fact, also able to induce male courtship behaviour in females when researchers artificially induce the male-specific expression. On the other hand, loss of male-specific fruitless in males causes the disappearance of all typical male courtship behaviour. This is further complicated by the fact that there are several different alleles (variants) of the fru gene, able to alter not only courtship, but sexual orientation as well. For example, males with certain mutant forms of fru display an enhanced form of homosexual behaviour — with males literally chasing each other and creating courtship “chains.”

In a study published this April in the journal Cell, researchers from Yale and Oxford University looked at the neuronal circuitry expressing the products of the fru gene in male and female flies, and investigated what happens when one activates this neuronal circuit in fru mutants of both sexes. The experiments showed that activating this circuit results in different responses in males and females, and that the responses are dictated by the version of the fru protein expressed by the animal. Thus, wild-type males and male-fru-expressing females can produce the wing movements and the courtship song, whereas wild-type females will move their wings and produce a sound, but not a courtship song. However, females will “sing out of tune,” because the ability to “sing in tune” is carried out by higher-order neurons that are probably not present or are inactivated in females.

Researchers have discovered methods of controlling neuronal activity, and thus manipulating fly courtship behaviour. Shining light on the neurons, which expressed a light-activated ion channel, artificially activated the neuronal circuitry. When positive ions are allowed into neurons by ion channels, the neurons depolarize and the depolarization wave, or action potential, moves all along the membrane of the neuron until it reaches the synapse, where it causes neurotransmitter release. All you need to do to activate a neuron in a controlled way is to make it express an ion channel that can be controlled.

How did researchers make sure that the channels were present in the neurons they are studying? They used a promoter specific to those neurons, so that the gene coding for the channels made protein only there. A promoter is a stretch of DNA that has a role in determining where the gene is expressed in the body — and what better promoter to study fru-expressing neurons than the fru promoter?

After the researchers had caused the right neuronal activity in the right places, using these methods, they observed the behaviour of single flies and couples. In a short video published by New Scientist about this study, the narrator says that “to make a female sing like a male, all you need to do is to turn on one gene, and chop her head off.” In fact, the head of female flies being observed was chopped off to facilitate the observations, as “singing” usually requires interaction with another fly, and it is intermittent — all of which is not helpful if you want to analyze the song of that one fly in the experiment. This also helped to separate the ventral ganglion from the head, increasing the success rate of the experiments, which would have otherwise been only around 1.7 per cent.

By now you might be wondering whether the fruitless gene is present in humans. While Wikipedia states that the fruitless gene is not present in mammals, an independent search shows that humans might have a homologue of the fly gene. The name of the homologue is ZBTB22, a gene predicted to be a transcription factor and a zinc finger DNA binding protein, just like fruitless is. Does this mean that it has the same function in humans? Well, given that we do not produce courtship songs using wings, and that sex determination is very different in humans and flies, it probably doesn’t.

Apart from the results and the media hype around fruitless flies, you might still be wondering what the scientific implications of the study are. What the results suggest is that behavioural differences between the sexes might not be necessarily due to differences in neural circuitry, but in the presence or absence of sex-specific regulators of such circuitry. This means that, although it is usually true that there are significant differences in the overall neuronal structure of males and females (with males sometimes having extra neurons dedicated to male-specific behaviour, the fact that some shared circuitry might also contribute to fundamental sex-specific behaviours is also something that needs to be considered.

But what are the more general philosophical implications of these studies on fruitless flies? Psychologists believe that our genes might be able to set a range of potential behaviours for each individual, and that interaction with the external environment determines where he or she sits in that range. According to behaviourism, a philosophy of psychology developed in the ‘60s, interaction with the environment (and possible conditioning that might be exerted by external conditions) is fundamental to the development of behaviour. One of the founders of behaviourism, John B. Watson, is known to have said that he could create, starting from any 12 infants, any behaviours and personalities he wanted, simply through conditioning and applying other behavioural techniques. While this extreme view is now generally discredited, it is still widely believed that, in general, genes alone are not able to completely determine behaviour, and therefore that no genotype is sufficient, on its own, to completely determine and elicit a given behaviour.

The results of the studies conducted on fruitless flies are a proof of principle that this is in fact possible in animals, and that at some certain behaviours can be completely determined by genetic makeup. There is a caveat to this statement: the gene needs to be necessary and sufficient to generate this behaviour, as is the case with fruitless and courtship behaviour in fruit flies. This does not imply that all behaviours in animals are strictly determined by their genetic makeup, but it does effectively prove that this can possibly happen, at least for some specific innate behaviours. Some might oppose that this does not extend to humans; after all, human behaviours are also affected by cultural norms, which means that humans might experience a higher pressure from their social environment to conform to certain behaviours.

However, we need to consider the fact that, at a cellular and molecular level, humans and flies are surprisingly similar. Studies on fruitless flies, from the ‘60s until now, have shown that a single gene, in this case a key behavioural regulator expressed in a sex-specific manner, can activate a neuronal circuit, and that such activation is sufficient and necessary to produce a specific behaviour, which is therefore already determined at a molecular and cellular level. This opens up the possibility that gene-specific regulation of neuronal pathways affecting or determining behaviour might be possible in other animals, and maybe even in higher organisms such as humans.

© 2008. The Peak Publications Society

Male fly image courtesy of Wikipedia Commons

Clyne, D., Miesenböck, G. (2008). Sex-Specific Control and Tuning of the Pattern Generator for Courtship Song in Drosophila.. Cell, 133(?), 354-363.

Related posts (generated)

• Female flies get turned on – by a light switch (3)

These studies on “fruitless” flies are some of the coolest studies on gender-specific behavior around. Although the BBC suggestively calls this a “mind-control sex swap“, this is definitely not a sex swap. One characteristic of fruitless flies is that their sexual characteristics are perfectly normal – the males are fertile, and so are the females, and there is no deformation of their bodies. So we are really not looking at the sex of the flies – but rather, at their gender. Which is what makes these studies so intriguing.

First of all, let me briefly explain how courtship works in Drosophila melanogaster. The male is the only one that can be said to display courtship behavior: when a male intends to court a female, it uses wing vibrations to produce a “courtship song”. This song can be identified because of the specific frequencies of the sounds emitted by the male. There are two “modes” of emission, known as the sine song, at ∼140–170 Hz, and the pulse song, constituted of brief and repetitive amplitude modulations in a range of ∼150–300 Hz. Females respond to this by allowing the male to copulate (or not; whether the male is successful depends on a variety of other factors, including winning “fights” with other males).

What about these fruitless (fru) flies? It was already known that fruitless encodes a gene product necessary for the determination of sex-specific courtship behavior in flies, and that the products of this gene exist in two main versions produced by alternative splicing: a male version and a female version. A previous study done in Barry Dickson’s lab showed that the male version is necessary for the determination of male courtship behavior and sexual orientation. The gene is in fact also able to induce male courtship behavior in females when spliced in a male-specific manner (causing the male version of the protein to be produced in females). On the other hand, loss of male-specific splicing in males causes the disappearance of all typical male courtship behavior.

This whole situation is complicated by the fact that there are several different alleles (variants) of the fru gene, able to alter not only courtship, but sexual orientation as well. For example, males with certain mutant forms of fru display an enhanced form of homosexual behavior. What do I mean by that? You might want to see for yourself in this video from one of the Dickson papers. Click on the image to go to the video, which gives a new dimension to the phrase “love train”.

Now we can get back to today’s study. Researchers working at Yale and Oxford have decided to look at the neuronal circuitry expressing the products of the fru gene in male and female flies, and to see what would happen when one activates this neuronal circuit in fru mutants of both sexes. The experiments showed that activating this circuit results in different responses in males and females, and that the responses are dictated by the version of the fru protein expressed by the animal. Thus, wild-type males and male-fru-expressing females can produce the wing movements and the courtship song; wild-type females will move their wings and produce a sound, but not a courtship song.

The neuronal circuitry was artificially activated by shining light on the neurons, which were made to express a light-sensitive ion channel.

What does this mean? When ions are allowed into neurons by ion channels, the neurons depolarize – and the depolarization wave moves all along the membrane of the neuron, till it reaches the synapse, where neurotransmitter release occurs. All you need to to do activate a neuron in a controlled way is to make it express an ion channel you can control. Ion channels need energy in the form of ATP to work, and therefore providing ATP can induce depolarization. You can then use a form of ATP linked to another compound: this compound makes the ATP “inactive”, but when you shine light on it, the “normal” ATP is released, and the ions channels open.

How do you make sure that the channels are present in the neurons you are studying? You use a promoter specific to those neurons – so that the gene coding for the channels makes protein only there. And what better promoter to study fru-expressing neurons…than the fru promoter? In fact, that is the one used in this study.

This short video summarizes the results of the study. I love when the narrator says that “to make a female sing like a male, all you need to do is to turn on one gene, and chop her head off”. It does give the wrong impression, doesn’t it? The head is chopped so that the female does not stop “singing”, as that behavior usually requires interaction with another fly, and it is intermittent. Which is not helpful if you want to analyze the song of that one fly in the experiment.

Now, apart from the results and the hype, you might still be wondering what are the implications of the study, from a more neurobiological point of view. What the results suggest is that behavioral differences between the sexes might not be necessarily due to differences in neural circuitry, but in the presence or absence of sex-specific regulators of such circuitry. Although it is usually true that there are differences in the overall neuronal structure of males and females (with males usually having extra neurons dedicated to male-specific behavior, which is something that is surely true of the nematode C. elegans), the fact that some shared circuitry might also be part to fundamental sex-specific behaviors is also something that needs to be considered.

As a last note, in case you are wondering: while some sources (which do not attach any citation to this statement) say that the fruitless gene is not present in mammals, I have run my own search, and it turns out that humans might have a homologue of the fly gene. The name of the homologue is ZBTB22 (which tells me this gene has not been well characterized), a transcription factor, just like fruitless is. Does this mean that it has the same function in humans? Well, given that we do not make courtship songs using wings, it probably doesn’t.

Curious facts: we have known about gay fly males for quite a while. In fact, the fru mutants were initially created because of X-ray radiation in 1963, and there is a 1989 paper describing the effects of the fruitless defects in males, and tracking them back to a chromosomal inversion, which must have probably been caused by DNA breaks and repairs following radiation exposure in the original strain. Nowadays people are looking at different mutated alleles of fru, not at the original version created through radiation exposure.

Citation

Clyne, D., Miesenböck, G. (2008). Sex-Specific Control and Tuning of the Pattern Generator for Courtship Song in Drosophila. Cell, 133(?), 354-363.

Male fly image courtesy of Wikipedia Commons

Related posts (generated)

• Gay love on the fly (0)