When it comes to genetic research, fruit flies take the spotlight. They are often used to study specific genes, and researchers who find new genes get the privilege of naming them. The best way to study a gene is to mutate it and see what happens when the gene’s function is lost. As a result, genes are usually named after the phenotype caused by the mutation. For example, one of the genes I work with is amnesiac. As you can probably guess, flies with a mutation in the amnesiac gene can’t form long-term memories!
This naming convention can often be very confusing for first-time fly students. One of the first genes we learn about is the white gene, which codes for eye color. Flies with a normal version of the white gene (usually written as w+ ) actually have red eyes, while flies with a mutation in the white gene ( w– ) have white eyes.
Over the years, fly scientists have amused themselves by choosing hilariously descriptive names for newly discovered genes. Here are some of my favorites (these are real scientific gene names):
Cheapdate– mutants have decreased resistance to the effects of alcohol
Hangover– mutants can’t develop a tolerance for alcohol
Dunce– mutants have learning deficits
Tinman– This gene regulates the development of a heart, so flies with a mutation in this gene can’t grow a heart. Named after the character in Wizard of Oz
Ken and Barbie – mutants do not develop external genitals
Swiss cheese – mutants have holes in their brain
I’m not dead yet – mutants live twice as long as normal flies, a reference to Monty Python and the Holy Grail
Groucho Marx – mutants develop an excess of facial bristles
Out Cold – mutants lose coordination at cold temperatures
Clumsy – mutants have trouble walking
Fly scientists have been getting away with choosing funny (and sometimes politically incorrect) gene names for decades. But over 75% of fruit fly genes are shared with humans, so not only would some of them eventually be found in humans, but they’d also be associated with a genetic disorder. And that’s where fly scientists can get themselves into trouble. For example, several years ago a gene named Hedgehog made headlines when it was found in humans.
The Hedgehog gene was discovered in 1980, and was given its name based on the observation that a mutation in this gene led to spiky-looking larvae. Three versions of this gene were later found in humans, one of which was named Sonic Hedgehog (SHH) after Sega’s video game character. Unfortunately, this gene has since been linked to a human developmental disorder called Holoprosencephaly, in which an SHH mutation leads to severe brain, skull, and facial defects. Doctors soon began to protest having to explain the joke name to patients with extreme medical conditions.
In 2006, the Human Genome Organization Gene Nomenclature Committee stepped in and changed some of the most offensively named genes. Another gene that was targeted was Lunatic Fringe, which was originally given its name because mutant flies had defects on the edges of their wings. In humans, however, mutations in the gene (which was renamed LFNG) leads a hereditary spinal defect called Jarcho-Levin Syndrome.
But despite these naming blunders, fruit fly research on these genes has and will continue to help in the search for treatments for these genetic disorders. And fly scientists haven’t stopped taking the opportunity to come up with fun names for newly discovered genes. Earlier this year, the Dickson fly lab published a report on “Moonwalker” flies: flies that couldn’t change direction and were permanently stuck walking backwards.
Video: The left side shows a fly walking normally. The right side shows a “moonwalking” fly unable to walk forwards.
How can we slow or even halt the steady march of aging? In a previous post, I reviewed a paper that asked “What causes aging?” (the prevailing hypothesis is that aging is caused by accumulating cell damage). Understanding why we age is important for developing ways to interfere with the process. But there are other ways to study aging in the hopes of one day developing the mythical “elixir of life”. A recent fruit fly paper published in Cell Reports by the Walker lab instead asked, “How does caloric restriction increase lifespan?”
Scientists discovered a long time ago that caloric restriction can extend lifespan in a wide range of animal models, including rats and fruit flies. But the diet is severe and leads to side effects including reduced energy levels and sensitivity to cold. Who wants to live longer in such misery? Decreased food intake itself can’t be the cause of increased lifespan (it does seem rather counterintuitive), so what is going on?
The authors found that activating a molecule called AMPK could increase lifespan just like dietary restriction, without all those miserable side effects. Normally, AMPK is activated when a cell needs more energy (such as when the animal isn’t eating enough calories). AMPK then triggers a process called macroautophagy, which means that components and molecules inside of cells are broken down and recycled for energy. This usually happens when the cell needs more energy than it is being provided, and the first things to go are old, unnecessary, and damaged cellular components. By artificially activating AMPK, the authors tricked the cells into breaking down more of this “cellular garbage”, slowing the accumulation of damage that leads to aging.
Perhaps the most exciting finding was that the authors could extend the lifespan of adult flies just by activating AMPK in the intestines. This manipulation slowed aging not just in intestinal cells, but also in brain tissue and even muscles. In fact, older flies even showed improved climbing ability. Although there is much work left to be done, these findings give hope that one day, aging can be slowed in adult humans just by swallowing a pill that activates AMPK in our gut.
Reference:
Ulgherait M., Michael Rera, Jacqueline Graniel & David W. Walker (2014). AMPK Modulates Tissue and Organismal Aging in a Non-Cell-Autonomous Manner, Cell Reports, 8 (6) 1767-1780. DOI: http://dx.doi.org/10.1016/j.celrep.2014.08.006
Why do we age? It’s more than just a philosophical question; it’s a puzzle that has frustrated scientists for decades. Currently, the most accepted hypothesis is that aging is the result of accumulated damage to our cells during our lifetime. “Accumulated damage” encompasses a variety of things that can go wrong, including DNA mutations, problems in the way molecules such as proteins are built, or abnormal interactions between molecules. Over time, the damage interferes with the body’s ability to maintain itself the way it used to, and the process we call “aging” occurs.
Click on the picture for a bigger version. Multiple external and internal factors can lead to the formation of free radicals and cell damage, which accelerates aging.
It sounds like a great hypothesis, but how can we prove it, and how can we measure accumulated damage? Damage can be caused by a number of factors, and the types of damage that can occur varies wildly between species and even between individuals of the same species. To make matters worse, exposure to certain environmental influences or toxins can accelerate aging. For example, overexposure to UV light from the sun can increase signs of aging in skin cells, and studies have shown that smoking can also accelerate aging. How can scientists study something with so much variability and uncertainty? A recent fruit fly paper published in eLife by the Gladyshev lab describes a new way to study damage accumulation. Instead of measuring the damage itself, they measure the byproducts of cellular metabolism as a proxy.
You see, even if we were able to reduce exposure to environmental toxins, we’d still get older. That’s because unfortunately, even our bodies are working against us. Our cells have metabolic processes, which are the life-sustaining chemical reactions that are needed to keep them alive and reproducing. The small molecules that are produced by these reactions (called metabolites) are usually important for the cells; they could be fuel for energy, signals for growth or reproduction, or even necessary for defenses and interactions with the environment. Unfortunately, these processes also create toxic byproducts such as reactive oxygen species (also known as free radicals). These byproducts cause damage and have often been associated with many age-related diseases such as cancer, heart disease, and Alzheimer’s disease.
To test the idea that accumulating metabolic byproducts can lead to aging, the authors of this study used a new method called “metabolite profiling” to measure the amount of metabolites in flies as they aged. (If you’re interested, the technique they used is called liquid chromatography mass spectrometry). They first found that the diversity of metabolites increases with age. This suggests that mistakes were being made during the metabolic reactions, causing new types of byproducts to appear that can damage cells. Additionally, a subset of metabolites accumulated with age, which may indicate that these byproducts are not being sufficiently cleaned up by maintenance processes in the cells. The authors found that many of these had previously been identified as damaging, directly confirming that accumulating toxic byproducts is correlated with aging.
Finally, the authors also used a calorie-restricted diet to extend the lifespan of a group of flies (although it’s not yet understood why, a severely restrictive diet can increase lifespan in many model animals, including mice and rats. It’s not recommended for humans, however, because there are unpleasant side-effects). Most interestingly, when the authors compared metabolite accumulation in normal flies versus the lifespan-extended flies, they showed that the metabolite accumulation was slower in the longer-lived flies, corresponding with the slower progression of aging.
So how can these findings help us? This paper supports the hypothesis that accumulation of damage leads to aging by showing that metabolites accumulate at a rate that corresponds with relative age in fruit flies. Because most species share the same cellular metabolic processes, these results are relevant to mammals. The next step will be to identify particular types of metabolites and determine how they contribute to aging in flies and mammalian models. The authors of this paper already did some of the legwork. They found that many of the metabolites that differed between the lifespan-extended group and the normal group of flies were associated with processes for using and storing energy from fats and proteins (not surprising considering the flies were on a strict diet). The authors suggest that changing these metabolic processes through diet may have compensated somehow for the accumulation of toxic byproducts. Future research may be able to expand upon these findings, and perhaps even figure out a way to interfere with these processes to slow or alter aging. I just hope I live long enough to see it!
Reference:
Avanesov A.S., Kerry A Pierce, Sun Hee Yim, Byung Cheon Lee, Clary B Clish & Vadim N Gladyshev (2014). Age- and diet-associated metabolome remodeling characterizes the aging process driven by damage accumulation, eLife, 3 DOI: http://dx.doi.org/10.7554/elife.02077
A study in 2012 found that approximately 7.2% of adults in the United States have an alcohol use disorder (a term that covers any person for whom their drinking causes distress or harm). That adds up to approximately 17 million Americans! Treatments for alcoholism, such as behavioral therapies or medications, can often be ineffective in the long-term due to changes that happen in the brain as a result of addiction. Improved treatment for alcoholism therefore requires an understanding of how and why addiction forms.
Figure 1. Genes can account for about 50% of the risk for alcoholism. Image by Addiction Blog
Research has shown that genes are responsible for about 50% of the risk for alcoholism, which can explain why alcoholism seems to run in families. Although genes alone don’t determine whether someone will become an alcoholic, they can influence a person’s sensitivity and tolerance for alcohol. Environmental or social interactions account for the remainder of the risk. A person with increased sensitivity to the stimulant effects of alcohol (increased energy, lowered inhibition) and/or decreased sensitivity to its depressant effects (motor impairment, sedation) might have a more pleasant experience with it, making them more likely to use it regularly. On the other hand, someone who finds the taste or side effects more aversive is unlikely to become addicted. For example, some people of Asian descent carry a gene variant that causes them to metabolize alcohol too fast, and they experience symptoms like flushing, nausea, and rapid heartbeat when they drink.
Studying the genes that increase the risk of alcoholism in animal models could lead to better preventive measures in at-risk families. Alcohol addiction also causes neuronal changes in the brain that can be studied in animal models to gain a better understanding of how the brain is changing and how to reverse it. Such an understanding will inevitably lead to better treatments for alcohol addiction.
Figure 2. Fruit flies can serve as a model for alcohol addiction. Drawing by the Heberlein Lab
Wait, isn’t this a fruit fly blog? How can they possibly have anything to do with alcohol addiction? You may have heard the old adage, “You catch more flies with honey than vinegar”, but in truth, fruit flies are more attracted to the smell of vinegar and fermenting fruit. Fruit flies like to feed off of the microbes (such as yeast) that are found in fermenting fruit, and they have evolved to develop a tolerance for the alcohol produced by fermentation. In fact, researchers have found that fruit flies even prefer to feed and lay their eggs on substances with about 4-5% alcohol—the concentration of an average beer.
One of the reasons fruit flies have learned to love alcohol is because it protects them from parasitic wasps, one of their most dangerous predators. Parasitic wasps inject their eggs into fruit fly larva. Although the larva’s immune system can sometimes resist the wasp growing inside it, most often the wasp larva eats the fly from the inside out, later bursting from it as a mature wasp. But while fruit flies have evolved a resistance to alcohol, the parasitic wasps have not. When a larva has been injected by one of these predators, it will consume almost toxic levels of alcohol in an attempt to kill off the parasite within it. In fact, female fruit flies that have detected the presence of parasitic wasps nearby will seek out the most alcohol-laden foods she can find before laying her eggs, giving her offspring their greatest chance to survive the predators.
But what happens when fruit flies are exposed to levels of alcohol not normally found in their natural environments? Stronger alcohol concentrations than what they have evolved to tolerate can actually be harmful to fruit flies. Does that mean fruit flies are smart enough to avoid such concentrations?
Figure 3. A fruit fly drinking alcohol from a capillary tube during a test for whether flies prefer food laced with alcohol. Photo by G. Shohat-Ophir, UCSD
Researchers have found that fruit flies seem to treat intoxicating levels of alcohol in the same way that some humans do—as a reward they just can’t get enough of. Fruit flies that have never been exposed to alcohol before will initially show a slight preference for it over a non-alcoholic food source, likely because it instinctively knows that alcohol signals food and safety. But with each exposure their preference for the alcohol-laden food gets stronger and stronger, despite its bitter taste and aversive side effects such as motor incoordination and sedation (sound familiar?). Eventually, these fruit flies show signs of alcoholism: they regularly drink until they’re “drunk”, build up a tolerance over time and drink more and more to compensate, and continue drinking despite increasingly dangerous side effects. They’ll keep drinking even if researchers add an aversive stimulus to the alcohol-laden food, such as a repulsive chemical or an electric shock. Fruit flies will also experience symptoms of withdrawal when the alcohol is taken away, and relapse to previous levels of drinking when it’s returned. Perhaps even more surprising is that drunk fruit flies lower their standards when looking for suitable sexual mates, and flies that have been sexually rejected will turn to alcohol to cope. These parallels between human and fruit fly drinking behavior are amazing! Scroll down to see a breakdown of the many ways fruit flies show signs of alcohol addiction.
These findings have led scientists to develop fruit flies as a model organism for alcohol addiction, because although fruit flies and humans may seem very different, many genes and cellular processes are shared between them (and in fact among most species!). Fruit flies are fantastic for genetic research, and could tell us a lot about genetic risks for alcoholism and why alcohol addiction forms. They can even be used to study the changes that occur in the brain as a result of addiction, since fruit flies exhibit many “alcoholic” behaviors.
What have fruit flies taught us so far? Fly scientists have already identified several genes that contribute to the risk of alcoholism (listed below). Despite their funny names, they could provide some very serious information about why some individuals have higher sensitivity or tolerance for alcohol and how these genes can be targeted by drugs to prevent or treat alcoholism.
Krasavietz – Researchers have found that fruit flies with a mutation in this gene are completely uninterested in alcohol. They showed that mutations in krasavietz reduced flies’ sensitivity to the “sedation” effect of alcohol, causing flies to quickly become sedated after intoxication, skipping all the fun stimulating side-effects.
Cheapdate – Flies with a mutation in this gene experienced increased sensitivity to alcohol, such that much lower doses were able to cause “intoxicated” behaviors.
Happyhour – A mutation in this gene causes flies to be less sensitive to the sedative effects of alcohol while maintaining normal sensitivity for the stimulating effects. A mutation in this gene in humans might increase the risk of addiction, because they will have a more pleasant experience when drinking and are more likely to drink regularly.
Hangover – Flies with a mutation in the hangover gene don’t develop a tolerance for alcohol. Instead of needing increased doses to achieve the same behavioral effects over time, the same dose always affects them the same way. Because increased consumption over time leads to dependence and addiction, human with a mutation in this gene may be at lower risk of alcoholism.
Researchers have also learned that a desire for alcohol in flies depends upon a certain chemical in the brain called neuropeptide F (NPF), which is very similar to neuropeptide Y (NPY) found in mammals. NPY signaling in mammals has been linked to stress, alcohol consumption, sexual motivation, and sugar satiety, among other things. In flies, reduced NPF levels led to increased alcohol intake. Sexual fulfillment was found to increase NPF levels, while sexual rejection decreased them. NPF levels could therefore indicate general “reward satisfaction”, and reduced levels causes flies to seek out something rewarding, whether it’s sex, drugs, or rock and roll. It is very likely that NPY in mammals plays the same role, which means manipulating NPY levels in humans could be a possible treatment for addiction.
Finally, fruit fly researchers have found that dopamine, a chemical that some neurons in the brain use for communication, was also involved in alcohol addiction. Dopamine has also been implicated in addiction in mammalian research because of the role it plays in the mammalian “reward system”, a brain region that gets hijacked in addiction. Further research in this area in flies may provide clues as to how dopamine signaling can lead to changes in “reward” structures.
No animal model will ever be a perfect model for alcoholism, because it is a largely a human phenomenon influenced by social, cultural, and cognitive factors. But animal models can be used to model important physical and behavioral facets of addiction, and to determine the genetic basis for withdrawal and tolerance. These findings will lead to the development of better medications for the prevention and treatment of alcoholism.
Fruit flies exposed to high levels of alcohol show behaviors that indicate they could be “tipsy” and/or “drunk”
Figure 4. The “booze-o-mat” is where fruit flies are placed in tubes with alcohol vapors to ensure consumption. Photo by the Heberlein Lab
While researchers sometimes provide fruit flies with alcohol by mixing it into their food, they can ensure consistency in their flies’ level of “drunkenness” by putting them in tubes filled with alcohol vapors so they breathe it in (Figure 4). This allows them to assess the behavioral effects of alcohol with less variation. At lower doses, the alcohol acts as a stimulant and increases their level of activity (comparable to increased energy and lowered inhibitions in humans). This is thought to be the rewarding effects of the alcohol. But increase the dose, and flies start showing signs of motor incoordination. They actually seem to be tipsy—they fall over, bump into each other and the walls, and have difficulties climbing. Even higher doses have a sedating effect.
Fruit flies prefer to consume alcohol, even if they don’t need it for nutritional purposes
Figure 5. Fruit flies are given a choice of capillary tubes containing either regular food or alcohol-laden food. Modified from Devineni & Heberlein, 2009
When fruit flies that have never experienced alcohol before are given a choice between non-alcoholic food and alcohol-laden food, they initially show only a slight preference for it. But the next time they are given a choice, they will overwhelming choose the food laced with alcohol. The flies will even consume enough alcohol to cause intoxication and alter its behavior as previously described. They will consume an intoxicating amount of alcohol time and time again with increased frequency, much like an alcoholic might.
Fruit flies will continue to consume alcohol even if they don’t like the way it tastes
Researchers have shown that while fruit flies like the way alcohol smells (it signals food and safety, after all), they don’t like the way it tastes. Nevertheless, just as humans consume alcohol despite its initially aversive bitter taste (and eventually develop a “taste for it”) fruit flies drink the alcohol anyway. Even when researchers associate alcohol with an aversive stimulus, such as giving the flies an electric shock whenever they drink or lacing the alcohol with a repulsive chemical called quinine, they will continue to drink up. This shows that flies are willing to overcome an aversive stimulus in order to consume alcohol.
Fruit flies develop a tolerance to alcohol and consume more and more each time to compensate
Like humans, fruit flies build up a resistance to the effects of alcohol after repeated exposure, which is known as tolerance. Flies that have been previously exposed to alcohol show an increasing tolerance to its effects over time, and will drink more and more each time to produce the same behavioral effects.
Fruit flies develop a physiological dependence on alcohol and experience symptoms of withdrawal
In humans, withdrawal symptoms include dysphoria, anxiety, cognitive impairment, and seizures. Clinically, these symptoms are a sign of alcohol dependence. After flies were chronically exposed to alcohol, they showed some of these same symptoms when it was taken away.
In one study, researchers showed that flies experiencing withdrawal had a lowered threshold for seizures and had more seizures than flies that had never been exposed to alcohol.
Researchers have also shown that fruit fly larva experience cognitive impairment as a symptom of withdrawal. They found that larva who drank too much had difficulties learning at first, but after chronic exposure they adapted and were able to learn almost as well as larva that were not exposed to alcohol. When the alcohol was taken away, their cognitive abilities were lost, and when the alcohol was returned, the larva also regained their learning abilities. These results suggest that the animals were dependent on alcohol not just physiologically, but also cognitively.
Fruit flies “relapse” after abstinence from alcohol
One characteristic of alcohol addiction is relapse, in which an individual will return to similar or greater consumption levels after a period of abstinence. In one study, fruit flies were chronically exposed to alcohol, followed by a period of abstinence. When the researchers provided them with alcohol again, the flies immediately began drinking at the same levels as before, without the gradual increase in preference that is seen when fruit flies are exposed for the first time.
Male fruit flies who have been sexually rejected turn to alcohol to cope
Researchers found that male fruit flies who had been unsuccessful in attempting to mate with a female drank more alcohol afterwards than males who were successful. They found that the desire to drink was dependent on levels of neuropeptide F (NPF), which were decreased after rejection but increased after successful mating. Alcohol consumption increased NPF levels again, suggesting that NPF acts as a “reward signal”. NPF is similar to mammalian neuropeptide Y (NPY), which has been linked to stress, anxiety, sexual motivation, alcohol consumption, and sugar satiety in mammals.
“Drunk” male fruit flies become hypersexual and lower their standards for suitable sexual mates
Figure 6. Drunk male fruit flies sometimes form a “courtship chain”, where male flies follow each other around trying to mate. Photo by Kyung-An Han laboratory, Penn State
Normally, male flies will only try to mate with females, but when they have been exposed to alcohol, they will not only step up their courting of females, but also even try to mate with other males. This effect got worse and worse with each exposure to alcohol. Unfortunately, just as humans have learned over the years, rates of successful mating actually decreased after getting tipsy. Even for fruit flies, getting drunk doesn’t necessarily lead to good sex.
References:
Milan N. & Todd A. Schlenke (2012). Alcohol Consumption as Self-Medication against Blood-Borne Parasites in the Fruit Fly, Current Biology, 22 (6) 488-493. DOI: http://dx.doi.org/10.1016/j.cub.2012.01.045
Kacsoh B.Z., N. T. Mortimer & T. A. Schlenke (2013). Fruit Flies Medicate Offspring After Seeing Parasites, Science, 339 (6122) 947-950. DOI: http://dx.doi.org/10.1126/science.1229625
Shohat-Ophir G., R. Azanchi, H. Mohammed & U. Heberlein (2012). Sexual Deprivation Increases Ethanol Intake in Drosophila, Science, 335 (6074) 1351-1355. DOI: http://dx.doi.org/10.1126/science.1215932
Lee H.G., Jennifer S. Dunning & Kyung-An Han (2008). Recurring Ethanol Exposure Induces Disinhibited Courtship in Drosophila, PLoS ONE, 3 (1) e1391. DOI: http://dx.doi.org/10.1371/journal.pone.0001391
Robinson B., Anna Kuperman & Nigel S. Atkinson (2012). Neural Adaptation Leads to Cognitive Ethanol Dependence, Current Biology, 22 (24) 2338-2341. DOI: http://dx.doi.org/10.1016/j.cub.2012.10.038
Devineni A.V. (2009). Preferential Ethanol Consumption in Drosophila Models Features of Addiction, Current Biology, 19 (24) 2126-2132. DOI: http://dx.doi.org/10.1016/j.cub.2009.10.070
… and all you ever wanted to know about fruit flies!
Have you ever wondered how fruit fly scientists perform experiments with these tiny insects every day? Since I work with fruit flies, I want to provide a glimpse of the day-to-day life of a fly researcher (imagine you’re a “fly on the wall” in our lab!). My project is to figure out how neurons in the memory system communicate with each other to store long-term memories. I dissect fruit fly brains and use fluorescence imaging to watch what neurons are doing in real-time, and I collaborate with other graduate students and postdocs in the lab who perform behavioral experiments. But what are the day-to-day activities that every fly scientist has to take care of?
Our stocks of flies are kept in incubators at a stable temperature and light cycle.
My day usually doesn’t start until 10am when the lights in our flies’ incubators turn on. Most of our flies are kept in these refrigerator-sized incubators at a tightly-controlled temperature of 25°C (77°F), which is a fruit fly’s favorite temperature. They’re also kept on a strict light cycle—12 hours of light starting at 10am followed by 12 hours of darkness, which is important for reducing variation in behavioral experiments.
Fly lines are kept in vials, which are stored on racks for easy stacking.
There are thousands of different fly lines, but I only have about 200 of them. Each fly line is kept in a small vial with food at the bottom (the yellow stuff, which is a thick gel of yeast, sugar, and cornmeal) and a cotton plug at the top to keep them from escaping. Each fly line has to be “flipped” to a new vial every couple of weeks after the hundreds/thousands of flies have finished making a mess of their current home.
A fly vial in use.
If you look closely, you can see pupal cases stuck to the walls of the vials. An adult fly can live for about a month at its optimal temperature, during which time a female can lay hundreds of eggs. The eggs hatch into larvae after approximately 24 hours, which spend about 6 days chewing through food and gaining strength. When the time is right, the larvae climb the sides of the vials and turn into pupae. After 4 motionless days, a new
Fruit flies go through four life stages: egg, larva, pupa, and adult. source
fly emerges from its pupal case. In total, it takes about 10 days after an egg is laid before a fly emerges. We can tell when a fly is about to come out because the pupa gets darker as the fly develops inside it. Within hours after emerging, an adult female fly is ready to mate and lay eggs.
Knocking out flies with CO2 in the vial prevents them from escaping
Flies are dumped onto a CO2 pad to keep them unconscious while they are being sorted.
Now I need to get flies out of some of those vials so I can use them for my experiments. Carbon dioxide (CO2) knocks flies unconscious, so we slide a syringe needle in through the cotton top of the vial and pump in some CO2 to knock them out. Then we can dump the flies out onto CO2 pads, which have a porous styrofoam top and CO2 flow underneath to keep the flies unconscious.
Wild flies (bottom) have bright red eyes, but lab flies (top) usually have white eyes until a gene with an eye color feature is added. source
For our experiments, we need to make sure our flies have the right genes or mutations. In my case, I need flies with a fluorescent marker in memory-specific regions of the brain so I can see them under a microscope. This usually means I need to collect flies with two added genes in their DNA: a “marker” gene and a “location” gene. I will need to sort through the flies to make sure they have both genes. Luckily, when a scientist makes a fly line with a new gene, they attach some DNA code for a physical feature to the gene. Usually, the feature is a change in the flies’ eye color. Although wild flies have bright red eyes, our lab flies are bred to have a mutation in the eye color gene so that they have white eyes.
Lab flies with an extra gene inserted into their DNA usually have orange eyes. The shade of orange varies, but darker colors can often mean more than one new gene is present. Photo by Weiwu Xie, source
Because our flies have no eye color, adding a gene with an eye-color feature makes these flies stand out among their white-eyed brethren. This way, we can sort through the flies and pick out the ones with colored eyes, most commonly orange. Flies with two genes have darker orange eyes because the color adds up, which is great for me.
We use a fine-tipped paintbrush to carefully sort through the flies without harming them.
I sort through the flies under a microscope using a paintbrush, and then brush the flies I need for my experiments into smaller vials to separate them.
“Fly morgues” are used to dispose of flies. They also conveniently double as fly traps.
There are, of course, tons of flies I don’t need. We can’t possibly save all the unnecessary flies, so they get dumped into a “fly morgue”. These are bottles filled with a mixture of water, ethanol, apple cider vinegar, and a drop of dish detergent. The funnel keeps them from escaping after we dump them in, and the detergent breaks the surface tension of the water so the flies can’t just skim across the surface. It’s a quick and painless death for our noble subjects, and at least they die swimming in their beloved vinegar. These also make great traps in the home to take care of fruit fly infestations. If you don’t have any vinegar, a splash of wine should do it.
A dissection microscope. A dish with wells for dissecting is shown underneath.
Now it’s time to prepare the flies for experiments. For some people in our lab, this means loading the live flies into machines that track their activity for sleep or memory studies. For others, such as myself, it means dissecting out the brains from individual flies (or in some studies, the larvae instead), and performing experiments on the brains instead of live animals. As you can imagine, it takes a pretty long time to learn how to dissect out the tiny brain from these insects. We use a microscope to see what we’re doing, and very carefully remove the shell of the fly head using fine-tipped tweezers.
A close-up “action shot” of a fly head being dissected. The head is being held with a pair of fine-tipped tweezers.
If you’re interested in seeing a dissection in action, check out this video from Jove, the Journal of Visualized Experiments.
A graphical representation of an adult fruit fly brain. Modified from Chiang et al, 2011
The final brain is too small to see by eye (though the most experienced among us can point out the tiny white dot in a drop of water). Fun fact: did you know that fruit flies don’t have blood like we do? They have an open circulatory system filled with “hemolymph”, which is a clear liquid filled
Click on the picture for a bigger version. The brain (circled in red) is floating in a solution. Can you spot it?
with nutrients for cells. We dissect brains in a solution that’s designed to mimic this hemolymph and keep the brain alive as though it was still inside the fly.
Close-up of the imaging set-up for dissected fly brains. Fresh hemolymph-like solution is being perfused in from the right to replace the old solution being vacuumed out on the left. A 60x magnification objective is shining a tiny beam of fluorescent light into the dish.
A structure in the fly brain called the “mushroom bodies” is fluorescing under a microscope. Photo by Jenett et al / CC-BY-2.0
Once I’ve dissected my brain, I pin it down in a dish for imaging. The brain-in-a-dish is placed under a microscope with a fluorescent light so that the fluorescent marker in the brain glows. During the experiments, I make sure that fresh hemolymph-like solution is flowing over the brain and being vacuumed out on the other side to keep the brain healthy, and sometimes I use this system to add in drugs which can affect activity in the neurons. This allows me to manipulate specific neurons and study how it affects communication between them.
In future “Fly Life” articles, I will go into detail about how specific experiments are performed, such as my imaging technique or the behavioral experiments used by my labmates. Until then…
Got any questions or want more details? Feel free to ask in the comments section!
Discussions about whether schools for children should start later have been making headlines recently, highlighting the importance of getting enough sleep at night. We all know how important sleep is for day-to-day performance—you’ve likely experienced firsthand how hard it can be to think and focus after a bad night’s sleep. Luckily, these effects are reversible: just get enough sleep for the next couple of nights and you’ll feel refreshed again. But can sleep deprivation have long-term, irreversible consequences in children?
Table 1. Human children require more sleep than adults. Data obtained from the National Sleep Foundation
Across multiple species, young animals need more sleep than adults. Although the purpose of sleep is not fully understood, researchers believe that the brain may use this time to repair itself, store new memories, and modify itself to stay current with recently learned skills or adapt to changes in the environment (a process known as plasticity). The brains of young animals are very plastic and are undergoing a lot of changes as they develop, and scientists have always suspected that increased sleep is necessary for normal brain development. In humans, the majority of brain growth occurs before the age of two, which is also the period of life with the highest amount of sleep. In a recent paper published in Science, the Sehgal lab studied the link between sleep and brain development using Drosophila melanogaster and found that loss of sleep in immature flies led to abnormal development in a fast-growing area of the brain and consequent behavioral problems in adult flies.
Just like humans and other mammals, fruit flies need a good night’s sleep to function normally during the day. The authors began their study by confirming that in flies, young animals also sleep more than adults. They then sleep deprived a group of flies by placing them in a shaking machine for two nights and measured their behavior three days later (a long time for a humble fruit fly!). Fly researchers prefer to study innate behaviors because they are instinctual instead of learned, suggesting that their underlying brain structure develops genetically rather than from experience (in these cases, it is thought that the “nature versus nurture scale” is tipped toward nature.) In this case, the authors studied “courtship behavior”, a measure of how well male flies can solicit female flies and successfully pass on his genes (so to speak). They found that flies that were sleep deprived when they were younger didn’t perform as well as flies that had gotten enough sleep. The authors showed that this behavioral abnormality was specifically caused by loss of sleep in young flies, because flies that were sleep deprived as adults performed normally three days later.
Figure 1. Sleep is required in young flies for normal development of fast-growing brain regions. Sleep deprivation during youth causes lack of growth in the VA1v, an important region for courtship behavior. Image modified from Murakami and Keene, 2014.
What happened in the brains of young sleep-deprived flies that led to their courtship inadequacy as adults? Previous research had already located the brain regions responsible for this innate behavior, so the authors knew where to start looking. They found that one of the regions (a structure called the VA1v) had not grown as much in flies that had been sleep-deprived when they were young. The VA1v is a very fast-growing region during development, and the authors showed that loss of sleep irreversibly slowed its growth. On the other hand, structures that do not undergo fast growth during development were normal. The authors concluded that sleep deprivation in young animals impairs brain development in fast-growing areas, resulting in irreversible behavioral abnormalities.
These results demonstrate just how important it can be to get enough sleep, especially as children. Research with human children already indicate that loss of sleep can have long-term effects on behavior, and the findings from this paper suggest that the consequences may not always be reversible if developing regions of the brain are affected. But what if children have a developmental or genetic disorder—such as a pediatric sleep disorder—that causes loss of sleep during this critical time period?
The authors also figured out the difference in young fly brains that caused them to sleep more than adults. Using the impressive set of genetic tools that Drosophila are famous for, they identified a small set of dopaminergic (DA) cells that behaved differently in young versus adult flies. Dopamine is a chemical in the brain that some neurons use to communicate with each other, and is already known to be important for the “be awake!” signal in several mammalian species and humans. The authors found that this particular set of DA neurons was less active in young flies than adults, while other DA neurons had the same activity level regardless of age. The neurons communicated with a sleep-related structure known as the dorsal fan-shaped body (dFSB). The authors found that in young flies, reduced activity in these DA neurons allowed the dFSB to be more active, causing the flies to sleep more. When the authors artificially activated the DA neurons, they found that young flies were unable to sleep and had behavioral problems three days later, while adult flies did not experience any long-term effects. This result matched the one they obtained when they sleep deprived young flies by shaking them, confirming that reduced activity in these neurons was responsible for the extra sleep in young flies.
How can knowing the circuit responsible for extra sleep in young flies help us humans? Dopamine plays the same role in causing wakefulness in mammals as it does in flies, and the dFSB is similar to known mammalian sleep-related structures (such as the VLPO). Researchers can use these findings as a starting point for identifying similar DA neurons in mammals. Eventually, scientists may be able to develop a treatment that acts on these neurons in children with sleep disorders, allowing them to get more sleep and ensure normal brain development during this critical period.
Figure 2. The circuit responsible for extra sleep in young flies. In young flies, a set of dopaminergic (DA) neurons is less active, allowing a sleep-related structure known as the dFSB to be more active and promote extra sleep. In adult flies, the DA neurons are more active and suppress dFSB activity, leading to relatively less sleep.
Original reference:
Kayser M.S., Yue Z. & A. Sehgal (2014). A Critical Period of Sleep for Development of Courtship Circuitry and Behavior in Drosophila, Science, 344 (6181) 269-274. DOI: http://dx.doi.org/10.1126/science.1250553
Parkinson’s disease is caused by the progressive death of neurons important for movement and results in symptoms such as shaking or rigidity in the limbs, slow movements, and difficulty walking. The primary treatment is a drug called L-Dopa, which compensates for the neuron loss but eventually becomes less effective as more and more neurons die off. Uncovering the cause of neuron death is necessary before treatments can be developed to stop it, and research in fruit flies has already begun to advance our understanding of the disease. A new study published in the open-access journal eLife by the Guo lab expands upon this previous work and uncovers a possible treatment option.
Although most cases of Parkinson’s disease have an unknown cause, about 10-15% are genetic. Two of the implicated genes, PINK1 and Parkin, have been well-studied in Drosophila melanogaster. Just as in humans, a mutation in these genes in fruit flies leads to neuron death and the loss of motor skills. But how? Fruit fly research has shown that PINK1 and Parkin maintain mitochondria, the structures inside cells that provide energy (think of them as little power plants). A single cell can have hundreds or even thousands of mitochondria, depending on its energy needs. Over time, mitochondria can become damaged and begin functioning abnormally. These rogue mitochondria must be broken down and replaced with healthy ones before their dysfunctional behavior can cause damage to the cell. This is where the proteins created by the PINK1/Parkin genes come in.
Figure 1. Pink1 latches onto mitochondria to determine whether or not they are healthy. If the mitochondrion is healthy, Pink1 is quickly removed. Otherwise, Pink1 binds to a passing parkin protein, triggering the destruction of the unhealthy mitochondrion. Image modified from Diedrich et al, 2011
PINK1’s job is to latch on to the surface of mitochondria and detect whether or not they are functioning normally. If the mitochondrion is fine, PINK1 gets removed and nothing else happens. On the other hand, if the mitochondrion has been damaged, PINK1 stays put and binds to a passing Parkin protein, which triggers the destruction of the offending mitochondrion. As you’ve probably guessed, a mutation in either the PINK1 or Parkin gene results in an accumulation of dysfunctional mitochondria and leads to cell death. This provides some explanation for why neurons are dying in patients in Parkinson’s disease.
PINK1/Parkin also maintain mitochondria in another way. Mitochondria regularly join with each other and then divide again to replenish their numbers. PINK1/Parkin helps to prevent damaged mitochondria from joining with healthy ones by breaking down a protein called mitofusin, which is responsible for joining mitochondria together. Cells with mutations in PINK1/Parkin have too much mitofusin, which means that damaged mitochondria can hurt the healthy ones by joining with them. To make matters worse, the ratio of joins to divisions is tightly controlled, so when the balance is tipped in favor of joining, big clumps of joined mitochondria begin to form.
The researchers in the Guo lab investigated other proteins involved in mitochondrial maintenance, searching for one that could compensate for mutations in PINK1/Parkin by preventing damaged mitochondria from joining with others and restoring the balance between joins and divisions. They turned their attention on MUL1, a protein that had previously been shown to interact with mitofusin. The authors discovered that adding extra MUL1 proteins into cells with a PINK1/Parkin mutation fixed the mitochondrial problems caused by the mutations! Drosophila neurons with a mutation in PINK1/Parkin have clumps of mitochondria, while normal cells show mitochondria evenly spread out. Incredibly, mutant cells with extra MUL1 protein showed a normal spread of mitochondria. Adding extra MUL1 into mutant cells somehow compensated for the PINK1/Parkin mutations and returned the balance between joins and divisions to normal.
How was the extra MUL1 able to reverse the over-joining of mitochondria? The authors answered this question by manipulating and measuring MUL1 and mitofusin levels in a variety of situations. They found that cells with a non-functional mutation in the MUL1 gene had clumps of mitochondria and too much mitofusin, just like in PINK1/Parkin mutants. On the other hand, normal cells with extra MUL1 protein actually had too little mitofusin and mitochondria that were small and fragmented, suggesting that the balance in these cells had shifted toward too much division. With further investigation, the authors realized that MUL1 protein was actually breaking down mitofusin just like Parkin.
So the addition of extra MUL1 protein can compensate for PINK1/Parkin mutations by breaking down the extra mitofusin, thus returning mitofusin levels back to normal and rebalancing the ratio of mitochondrial joins to divisions. But this research was in fruit flies, so how do we know this will be useful for humans? The authors took their research a step further by demonstrating that MUL1 has the same function in mouse neurons and HeLa cells (human cells), and that extra MUL1 in these models still compensates for PINK1/Parkin mutations.
This is a fantastic finding, but unfortunately it doesn’t mean we’re ready to give MUL1 pills to Parkinson’s patients and cure the disease. The authors introduced extra MUL1 proteins genetically using a method called gene overexpression (check out this Wikipedia article on gene expression for more information). Basically, the authors made the cells produce their own extra MUL1, but a possible treatment would require developing and testing a drug that either forces cells to start making more MUL1 or adds MUL1 directly. Second, MUL1 doesn’t play a role in targeting and destroying damaged mitochondria. This means that while extra MUL1 could help to prevent clumps of mitochondria (which would have spread the damage from unhealthy ones faster), it can’t actually remove damaged mitochondria. So this treatment would not be able to completely stop the accumulation of dysfunctional mitochondria. But there is still hope! This option will be better than our current treatments because it could slow the progression of cell death instead of simply compensating for the loss. And in the future, research in this area will build upon these findings to develop an even better drug.
Figure 2. An increase in MUL1 levels can compensate for loss of PINK1/Parkin and maintain normal mitofusin (mfn) levels (A). A loss of either MUL1 (B) or PINK1/Parkin (C) alone causes an increase in mfn levels. Cells with a loss in both PINK1/Parkin and MUL1 show an even greater increase in mfn levels (D). Image modified from Yun et al, 2014
Yun J., Huan Yang, Michael A Lizzio, Chunlai Wu, Zu-Hang Sheng & Ming Guo (2014). MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin, eLife, 3 DOI: http://dx.doi.org/10.7554/elife.01958
Diedrich M., Grit Nebrich, Andrea Koppelstaetter, Jie Shen, Claus Zabel, Joachim Klose & Lei Mao (2011). Brain region specific mitophagy capacity could contribute to selective neuronal vulnerability in Parkinson’s disease, Proteome Science, 9 (1) 59. DOI: http://dx.doi.org/10.1186/1477-5956-9-59
Parkinson’s disease is the second most common neurodegenerative disorder, and patients experience primarily movement-related symptoms including shaking and rigidity in their limbs, slow movements, and difficulty walking, all of which progressively worsen over time. It was formally recognized as a disease in 18171, but didn’t receive much attention until it was given its name in 1861. Parkinson’s disease was not very well understood, and there were no real treatments for about 100 years. Then, in the 1960s researchers discovered that Parkinson’s patients had low levels of dopamine, a chemical in the brain that some neurons use for communication. This finding led to the use of L-Dopa (also known as levodopa) as a treatment. L-Dopa is taken orally and can reach the brain, where it is converted to dopamine2. Now approximately 40 years later, L-Dopa is still the primary treatment for Parkinson’s disease.
Figure 1. Parkinson’s patients show a decrease in dopamine levels in the brain. source
The problem is that L-Dopa doesn’t work forever. Since the 1960s, we have learned that Parkinson’s disease is caused by progressive damage and eventual loss of dopaminergic (DA) neurons (these are the ones that release the dopamine), most severely in an area of the brain important for movement known as the basal ganglia3. As more and more DA neurons become damaged and die off, L-Dopa loses its ability to compensate and the symptoms start to come back. To make matters worse, L-Dopa has a number of side effects that need to be treated with yet more drugs. Of course, L-Dopa is currently very necessary for treating Parkinson’s disease and can give patients an extra 5-15 years of quality symptom-free life. Even as L-Dopa’s effectiveness begins to decrease, it’s still better than no treatment at all. But newer and better treatments need to be developed.
Unfortunately, we can’t fix the disease until we understand what’s causing it. Why are the DA neurons dying? How can we stop it and then reverse the damage? We’ve learned just about everything we can from (ethical) research in humans. It’s time to bring in the model animals! The humble fruit fly Drosophila melanogaster has emerged as a particularly important model because while most cases of Parkinson’s disease occur randomly, about 10-15% of cases are due to inherited genetic mutations. And for genetic research, flies are our best bet.
Why study the genetic mutations if only 10-15% of cases are caused by them? The easiest answer is because we can introduce mutations into the same genes in fruit flies to find out what those genes do. If researchers can figure out what’s causing damage to the DA neurons in the genetic cases, those findings can be directly applied to the random cases. So even if we don’t yet know what caused them, treatments that protect DA neurons and compensate for their damage should still work to improve symptoms in both genetic and random cases of Parkinson’s disease.
Since the late 1990s, mutations in five genes have been found to lead to inherited forms of Parkinson’s disease in some families. Each was given a cryptic name: SNCA4, Parkin5, PINK16, DJ-17, and LRRK28-9. Once those genes were identified in human patients, researchers were able to mutate the same genes in animal models to figure out what they do and how their failure leads to Parkinson’s disease. Luckily for us, fruit flies already have four of those genes, so it was relatively easy to make a fly line with a mutation in them. The remaining gene, SNCA, can still be studied by introducing the human mutation into flies using a binary expression system (UAS/GAL4).
Figure 2. alpha-synuclein proteins clump to form masses called Lewy bodies. Figure depicts magnified image of a Lewy body surrounded by neurons in the substantia nigra (a part of the basal ganglia) in a patient with Parkinson’s disease. Photo by Suraj Rajan / CC BY-SA 3.0
So what have we learned so far from our winged friends? An overview of fruit fly research for each gene is listed below, but if you just want the punchline, here it is: Parkin,PINK1, DJ-1, and LRRK2 are all involved in maintaining and/or protecting mitochondria, which are structures inside cells that create the fuel the cell uses as energy (think of them as little power plants). When any of these four genes is mutated, mitochondria begin to function abnormally and are more sensitive to damage from environmental stressors such as toxins. As more and more damage accumulates, DA neurons begin to die off, which may explain why Parkinson’s disease is progressive and usually begins later in life. On the other hand, although the role of normal SNCA is still unknown, a mutated SNCA gene creates masses in the brain called Lewy bodies that may cause damage to DA neurons. Although it’s not fully understood why mutations in these genes affect DA neurons more severely than other neurons in the brain, it is thought that DA neurons may simply be more sensitive to environmental toxins.
Okay, but what about the random cases? While the cause of most of the random cases of Parkinson’s disease remains a mystery, results from research in the genetic cases can give us a clue. It is currently thought that this form of Parkinson’s disease is caused by a combination of factors such as accumulated damage and genetic mutations with age, exposure to environmental toxins such as pesticides, and genetic predisposition (meaning that there may be genes that don’t directly cause Parkinson’s disease, but may increase your risk if exposed to certain environmental triggers).
Research in fruit flies has definitely improved our understanding of the underlying causes of Parkinson’s disease, which will ultimately lead to the development of better treatments. Instead of simply compensating for reduced dopamine levels, future treatments may target the Lewy bodies or bolster the cell’s protective mechanisms for mitochondria. Treatments such as these will have longer lasting effects because they could potentially prevent further loss of DA neurons. And fruit flies aren’t our only hope. Many of the findings described above have already been used for designing studies in mammalian models, and research in mammals has also led to important discoveries not mentioned here.
Findings in fruit flies specific to each Parkinson-related gene:
Parkin/PINK1: I grouped these two mutations together because fruit fly researchers have recently discovered that these genes play similar roles in neurons10-12. PINK1’s protein actually interacts with Parkin’s protein to maintain and protect mitochondria. As a result, a mutation in either PINK1 or Parkin causes mitochondrial defects and increases sensitivity to environmental stress from toxins. The accumulation of damage leads to death in DA neurons and, of course, the resulting impairments in movement as seen in Parkinson’s patients. Further understanding of how PINK1 and Parkin work will allow researchers to develop drugs to compensate for lost function in these genes.
SNCA: Many Parkinson’s patients develop dense masses in their brains called Lewy bodies, which are formed when large molecules called proteins accumulate abnormally and bind to each other. Lewy bodies are primarily made up of the protein alpha-synuclein bound to various other proteins (alpha-synuclein is made from the instructions in the SNCA gene). But why is alpha-synuclein clumping together? Researchers introduced the mutated version of the human SNCA gene in fruit flies and found that mutant flies showed progressive DA neuron death and loss of motor skills, just like the symptoms in human Parkinson’s patients13. And of course, they found clumps of protein. Studies in this fly model are now focused on understanding why the mutated version of alpha-synuclein clump together and cause DA neuron death. Once these questions have been answered, researchers can develop treatments that either prevent formation of Lewy bodies, break them down, or prevent them from damaging neurons. Interestingly, researchers have found that when they add extra normal PINK1 or Parkin protein in this SNCA mutant, the extra PINK1 or Parkin actually helps to protect against DA neuron death14-15. So, another treatment option for Parkinson’s patients with Lewy bodies may be to stimulate extra PINK1 or Parkin protein production as a protective measure.
DJ-1: Researchers have found that fruit flies with a mutation in DJ-1β (flies actually have two versions of this gene, and DJ-1β is similar to the human version) are more sensitive to environmental toxins, demonstrating that this gene plays a protective role16. Flies with this mutant gene also showed reduced lifespan and locomotor defects, as seen in Parkinson’s patients17. Finally, mutant DJ-1 protein resulted in abnormal mitochondrial function, suggesting that this gene, like PINK1 and Parkin, is necessary for normal functioning in mitochondria18.
LRRK2: Mutations in LRRK2 are likely the most common genetic cause of Parkinson’s disease in humans, but it has so far been the most inconsistent in fruit flies, making it a difficult gene to study. Researchers have performed several kinds of genetic manipulations, including completely knocking-outLRRK2 function, introducing the mutated version of the human gene, or mutating it in a way that changed its function instead of making it completely non-functional. Some researchers found that the mutations led to DA neuron death and severely impaired movement, some researchers found no effect, and others found a result somewhere in the middle19. A few important reliable discoveries have come out of research in this gene, however. First, all of the mutants were more sensitive to various environmental toxins. Second, researchers found that LRRK2 proteins interact with mitochondria, suggesting that it, like PINK1 and Parkin, plays a role in maintaining and/or protecting these structures. These findings support an increasingly convincing conclusion that Parkinson’s disease may be caused by mitochondrial defects or damage that leads to death in DA neurons.
Blandini F., Cristina Tassorelli & Emilia Martignoni (2000). Functional changes of the basal ganglia circuitry in Parkinson’s disease, Progress in Neurobiology, 62 (1) 63-88. DOI: http://dx.doi.org/10.1016/s0301-0082(99)00067-2
<Polymeropoulos M.H. (1997). Mutation in the -Synuclein Gene Identified in Families with Parkinson’s Disease, Science, 276 (5321) 2045-2047. DOI: http://dx.doi.org/10.1126/science.276.5321.2045
Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y., Minoshima S., Yokochi M., Mizuno Y. & Shimizu N. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism., Nature, PMID: http://www.ncbi.nlm.nih.gov/pubmed/9560156
Valente E.M. (2004). Hereditary Early-Onset Parkinson’s Disease Caused by Mutations in PINK1, Science, 304 (5674) 1158-1160. DOI: http://dx.doi.org/10.1126/science.1096284
Bonifati V. (2003). Mutations in the DJ-1 Gene Associated with Autosomal Recessive Early-Onset Parkinsonism, Science, 299 (5604) 256-259. DOI: http://dx.doi.org/10.1126/science.1077209
Paisán-Ruı́z C., E.Whitney Evans, William P. Gilks, Javier Simón, Marcel van der Brug, Adolfo López de Munain, Silvia Aparicio, Angel Martı́nez Gil, Naheed Khan & Janel Johnson & (2004). Cloning of the Gene Containing Mutations that Cause PARK8-Linked Parkinson’s Disease, Neuron, 44 (4) 595-600. DOI: http://dx.doi.org/10.1016/j.neuron.2004.10.023
Zimprich A., Petra Leitner, Peter Lichtner, Matthew Farrer, Sarah Lincoln, Jennifer Kachergus, Mary Hulihan, Ryan J. Uitti, Donald B. Calne & A.Jon Stoessl & (2004). Mutations in LRRK2 Cause Autosomal-Dominant Parkinsonism with Pleomorphic Pathology, Neuron, 44 (4) 601-607. DOI: http://dx.doi.org/10.1016/j.neuron.2004.11.005
Clark I.E., Changan Jiang, Joseph H. Cao, Jun R. Huh, Jae Hong Seol, Soon Ji Yoo, Bruce A. Hay & Ming Guo (2006). Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin, Nature, 441 (7097) 1162-1166. DOI: http://dx.doi.org/10.1038/nature04779
Park J., Sungkyu Lee, Yongsung Kim, Saera Song, Sunhong Kim, Eunkyung Bae, Jaeseob Kim, Minho Shong, Jin-Man Kim & Jongkyeong Chung & (2006). Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin, Nature, 441 (7097) 1157-1161. DOI: http://dx.doi.org/10.1038/nature04788
Yang Y., Y. Imai, Z. Huang, Y. Ouyang, J.-W. Wang, L. Yang, M. F. Beal, H. Vogel & B. Lu (2006). Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin, Proceedings of the National Academy of Sciences, 103 (28) 10793-10798. DOI: http://dx.doi.org/10.1073/pnas.0602493103
Bell J. & Brian E. Staveley (2008). Pink1 suppresses α-synuclein -induced phenotypes in a Drosophila model of Parkinson’s disease , Genome, 51 (12) 1040-1046. DOI: http://dx.doi.org/10.1139/g08-085
Haywood A.F.M. & Staveley B.E. (2004). Parkin counteracts symptoms in a Drosophila model of Parkinson’s disease., BMC neuroscience, PMID: http://www.ncbi.nlm.nih.gov/pubmed/15090075
Yang Y., Md. E. Haque, Y. Imai, J. Kosek, L. Yang, M. F. Beal, I. Nishimura, K. Wakamatsu, S. Ito & R. Takahashi & (2005). Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling, Proceedings of the National Academy of Sciences, 102 (38) 13670-13675. DOI: http://dx.doi.org/10.1073/pnas.0504610102
Meulener M., Cecilia E. Armstrong-Gold, Patrizia Rizzu, Peter Heutink, Paul D. Wes, Leo J. Pallanck & Nancy M. Bonini (2005). Drosophila DJ-1 Mutants Are Selectively Sensitive to Environmental Toxins Associated with Parkinson’s Disease, Current Biology, 15 (17) 1572-1577. DOI: http://dx.doi.org/10.1016/j.cub.2005.07.064
Hao L.Y. & N. M. Bonini (2010). DJ-1 is critical for mitochondrial function and rescues PINK1 loss of function, Proceedings of the National Academy of Sciences, 107 (21) 9747-9752. DOI: http://dx.doi.org/10.1073/pnas.0911175107
Muñoz-Soriano V. (2011). Drosophila Models of Parkinson’s Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies, Parkinson’s Disease, 2011 1-14. DOI: http://dx.doi.org/10.4061/2011/520640
General references and reviews:
Muñoz-Soriano V. (2011). Drosophila Models of Parkinson’s Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies, Parkinson’s Disease, 2011 1-14. DOI: http://dx.doi.org/10.4061/2011/520640
You’ve probably heard the term “genome” before, which refers to the set of genes in an organism. Biology has a lot of buzzwords for describing what’s going on inside cells, and one of the recent ones is “interactome”. The word “interactome” refers to all of the interactions that are occurring between proteins, the large molecules that do most of the work inside your cells. Proteins are built from their corresponding gene’s instructions, and they drive cellular processes by interacting with each other. For example, when a receptor protein on the cell membrane is triggered—such as by recognizing an invading virus—it temporarily binds to another type of protein, which interacts with yet another protein and so on, until the cell’s immune response is fully activated and self-defense proteins are released to deal with the invaders. This series of protein interactions is called a “signaling cascade”.
As you might have guessed, if one of the proteins in a signaling cascade isn’t interacting properly, it leads to problems which can be the basis of diseases, such as Alzheimer’s disease, ALS, or cancer. As a result, it is very important to understand how proteins interact, especially when mutations can lead to human diseases. Unfortunately, a single protein can have dozens or even hundreds of binding partners. To make matters worse, it is thought that proteins have cell-specific interactions, which means that a given protein may interact with one set of proteins in one type of cell, and a different set of proteins in another cell type. However, current techniques for studying a protein’s binding partners use whole brains or brain structures consisting of many different types of cells and cannot distinguish among them.
A recent paper1 published in the Griffith lab addresses this problem by taking advantage of the genetics tools developed in Drosophilamelanogaster. They created a fly line with a mutation in the gene for the protein of interest, and then used a binary expression system (UAS/GAL4) to reintroduce normal versions of the protein into specific subsets of cells. The authors then followed up with current techniques for studying the protein’s interactions, but they knew that the binding partners were specific to the cells they were interested in.
To demonstrate their new technique, the authors studied a protein called CASK. In mammals, CASK is important for signaling between neurons and is implicated in two human developmental disorders. In flies, CASK is present in almost all neurons, and CASK mutants have problems with locomotion and learning. Researchers already knew that different types of neurons were responsible for each of these behaviors, and thought that CASK may have different interactions based on the type of cell. To test this hypothesis, the authors used a fly line with a non-functioning mutation in the CASK gene, and then reintroduced normal versions of the protein in three different types of neurons. They were then able to use current identification techniques (check out this link on mass spectrometry, if you’re interested) to determine which proteins had bound to CASK in each of the three lines and compare them to each other (as well as to a fly line where they had reintroduced CASK in all neurons). They found that while there were many proteins that interacted with CASK in all of the neuron subtypes, each group also had a set of unique interactions.
So what’s the bottom line? How can the findings in this paper help us? Because abnormal protein interactions are the basis of many human diseases, the only way to treat them is to determine which proteins are involved and understand their function. Only then we would know how to fix the problem. Unfortunately, while many of those interactions are specific to certain types of cells, current techniques require researchers to investigate the protein’s binding partners from many cell types. This creates unnecessary complexity because only specific populations of neurons are affected in some diseases. For example, in Parkinson’s disease, a type of cell known as dopaminergic neurons are most seriously affected. Researchers are studying proteins known to be involved in the disease, but may benefit from being able to limit the interactions to those that occur within dopaminergic neurons. Using fruit flies, the Griffith lab developed a technique for uncovering cell-specific differences in a protein’s interactions. In the future, researchers will be able to use Drosophila as a model organism for studying cell-specific protein interactions involved in human disease, and this technique may even be modified for use in mammalian systems.
Mukherjee K., Bethany L. Christmann & Leslie C. Griffith (2014). Neuron-specific protein interactions of Drosophila CASK-β are revealed by mass spectrometry, Frontiers in Molecular Neuroscience, 7 DOI: http://dx.doi.org/10.3389/fnmol.2014.00058
In my previous post, I described how Drosophila melanogaster serves as an important and relevant model organism for biological research. But how is fruit fly research actually helping us to better understand ourselves? In my future “Translational Findings” posts, I will talk about how fruit flies are furthering our understanding of a specific human-related issue. These will include diseases such as Parkinson’s or Alzheimer’s disease, developmental and genetic disorders such as autism or Down syndrome, and other human concerns like addiction, sleep, or aging.
In this first Translational Findings post, however, I would like to give an overview of the history of fruit fly research and how it has already contributed to human health. Fruit flies have been used as a model organism for over a century, so the list is long. To narrow it down, I will focus on describing the important findings that led to Nobel Prizes: four of them since Thomas Hunt Morgan published the first scientific paper using Drosophila melanogaster in 19101!
Figure 1. The fly on the top has a mutation that causes white eyes. The fly on the bottom has normal red eyes. source
The first Nobel Prize was awarded to Thomas Hunt Morgan himself in 1933. He studied heredity and was interested in understanding how physical traits were passed down through generations. Morgan began by searching for visible variations among fruit flies so he could determine how those traits were inherited. Finally, he found white-eyed flies among a stock of normal red-eyed flies (Figure 1). He and his students began studying the pattern of inheritance for the white-eyed trait, and they later found other mutations to study as well. Their findings led to a radical new theory of heredity which suggested that genes (the pieces of DNA that contain the information for the traits) are carried in a linear arrangement on chromosomes, and these chromosomes are passed down through generations. Their findings showed the physical mechanism for genetic inheritance and are now considered the foundation of modern genetics.
The second Nobel Prize was awarded to Hermann Müller, one of Morgan’s students. After leaving Morgan’s lab, Müller began researching methods for inducing mutations in fruit flies instead of waiting for them to occur spontaneously. In the 1920s, he made a breakthrough when he noticed a connection between radiation and lethal mutations and, in 1927, published a paper demonstrating that X-rays damaged chromosomes and caused genetic mutations2. Although the public was beginning to realize that radiation was dangerous (Marie Curie died in 1934 as a result of her own research), this was the first evidence of specific harmful effects. Müller began publicizing the dangers of radiation soon after, and earned a Nobel Prize in 1946 for his work.
In 1995, the third Nobel Prize for fruit fly research was shared by Christiane Nüsslein-Volhard, Eric Wieschaus, and Ed Lewis. Using recently developed techniques that allowed DNA to be more easily manipulated (such as X-ray induced mutations), these scientists screened thousands of mutant flies and identified several genes responsible for development in Drosophila melanogaster3. Their research paved the way for understanding how multicellular organisms develop from single cells, and showed that development is genetically controlled. Shortly after their discoveries, studies in other species found closely related developmental genes in vertebrates, confirming an evolutionary link between fruit fly and human biology.
The final Nobel Prize was award to Jules Hoffmann, Bruce Beutler, and Ralph Steinman in 2011 for their research in immunity. Humans have two methods for defending against infections: innate immunity, which is inherited, and adaptive immunity, which responds to invaders and “learns”. Hoffman’s research in fruit flies showed that a gene called Toll was important for the fly’s innate immune system. He found that the Toll gene contained instructions for a receptor responsible for recognizing certain bacterial and fungal infections and triggering an immune response4. Beutler later found related “Toll-like” receptors with the same function in mammals, demonstrating that this innate immunity control mechanism is shared across species through evolution. A few years later, Steinman showed that Toll-like receptors activate the adaptive immune system in mammals as well.
Fruit fly research has already made huge contributions to understanding human biology, and it shows no signs of stopping. In today’s research environment, research in flies has gone beyond the genetic research it founded and has moved into more complex issues such as disease and behavior. Which new major contribution will earn this little insect its fifth Nobel Prize?
Morgan TH (1910). SEX LIMITED INHERITANCE IN DROSOPHILA. Science (New York, N.Y.), 32 (812), 120-2 PMID: 17759620
Muller HJ (1927). ARTIFICIAL TRANSMUTATION OF THE GENE. Science (New York, N.Y.), 66 (1699), 84-7 PMID: 17802387
Nüsslein-Volhard C, & Wieschaus E (1980). Mutations affecting segment number and polarity in Drosophila. Nature, 287 (5785), 795-801 PMID: 6776413
Lemaitre B, Nicolas E, Michaut L, Reichhart JM, & Hoffmann JA (1996). The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell, 86 (6), 973-83 PMID: 8808632
Image modified from Nüsslein-Volhard and Wieschaus, 1980.
Drawing by the talented artist at Drosophila Drawings.
Figure 1. Genes can account for about 50% of the risk for alcoholism. Image by 
Figure 2. Fruit flies can serve as a model for alcohol addiction. Drawing by 
Figure 3. A fruit fly drinking alcohol from a capillary tube during a test for whether flies prefer food laced with alcohol. Photo by G. Shohat-Ophir, UCSD
Figure 4. The “booze-o-mat” is where fruit flies are placed in tubes with alcohol vapors to ensure consumption. Photo by 
Figure 5. Fruit flies are given a choice of capillary tubes containing either regular food or alcohol-laden food. Modified from 
Figure 6. Drunk male fruit flies sometimes form a “courtship chain”, where male flies follow each other around trying to mate. Photo by Kyung-An Han laboratory, Penn State
Our stocks of flies are kept in incubators at a stable temperature and light cycle.
Fly lines are kept in vials, which are stored on racks for easy stacking.
A fly vial in use.
Fruit flies go through four life stages: egg, larva, pupa, and adult. 
Knocking out flies with CO2 in the vial prevents them from escaping
Flies are dumped onto a CO2 pad to keep them unconscious while they are being sorted.
Lab flies with an extra gene inserted into their DNA usually have orange eyes. The shade of orange varies, but darker colors can often mean more than one new gene is present. Photo by Weiwu Xie, 
We use a fine-tipped paintbrush to carefully sort through the flies without harming them.
“Fly morgues” are used to dispose of flies. They also conveniently double as fly traps.
A dissection microscope. A dish with wells for dissecting is shown underneath.
A close-up “action shot” of a fly head being dissected. The head is being held with a pair of fine-tipped tweezers.
A graphical representation of an adult fruit fly brain. Modified from 
Table 1. Human children require more sleep than adults. Data obtained from the 
Figure 1. Sleep is required in young flies for normal development of fast-growing brain regions. Sleep deprivation during youth causes lack of growth in the VA1v, an important region for courtship behavior. Image modified from 
Figure 2. The circuit responsible for extra sleep in young flies. In young flies, a set of dopaminergic (DA) neurons is less active, allowing a sleep-related structure known as the dFSB to be more active and promote extra sleep. In adult flies, the DA neurons are more active and suppress dFSB activity, leading to relatively less sleep.
Figure 1. Pink1 latches onto mitochondria to determine whether or not they are healthy. If the mitochondrion is healthy, Pink1 is quickly removed. Otherwise, Pink1 binds to a passing parkin protein, triggering the destruction of the unhealthy mitochondrion. Image modified from 
Figure 2. An increase in MUL1 levels can compensate for loss of PINK1/Parkin and maintain normal mitofusin (mfn) levels (A). A loss of either MUL1 (B) or PINK1/Parkin (C) alone causes an increase in mfn levels. Cells with a loss in both PINK1/Parkin and MUL1 show an even greater increase in mfn levels (D). Image modified from 
Figure 1. Parkinson’s patients show a decrease in dopamine levels in the brain. 
Figure 2. alpha-synuclein proteins clump to form masses called Lewy bodies. Figure depicts magnified image of a Lewy body surrounded by neurons in the substantia nigra (a part of the basal ganglia) in a patient with Parkinson’s disease. 
Figure 1. The fly on the top has a mutation that causes white eyes. The fly on the bottom has normal red eyes. 