Drosophila Workers Unite! Lab Manual PDF

Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein 1 2 B y M i c he le M a rk s...

Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein 1 2 B y M i c he le M a rk s tein  Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein Illustrations by Kristopher Kolbert Photography by Jonathan DiRusso, Kristopher Kolbert, and Michele Markstein This book is dedicated to all my students— past, present, and future. © 2018 Michele Markstein Published under a Creative Commons Attribution-NonCommercial (CC-BY- NC) International License, version 4.0. This book is published under a CC BY-NC license. Under this license, authors retain ownership of the copyright in their work, but allow anyone to download, reuse, reprint, modify, distribute, and/or copy the article, as long as the original authors and source are cited and the intended use is not for commercial purposes. Michele Markstein owns the copyright of the text, photographs, and book as a whole; Michele Markstein and Kristopher Kolbert jointly own the copyright to the illustrations. Title and Description of Primary Image: Drosophila Workers Unite! This homage to “Rosie the Riveter” celebrates workers, women, and the model organism Drosophila melanogaster. Contact author at: [email protected] 3 1  Ack n owle dgemen ts Are you new to Drosophila? I am grateful to my graduate and postdoctoral mentors, former labmates, and the students in my own lab for making this book possible. In particular, Then this book is for you! I would like to thank Kristopher Kolbert, a former undergraduate in my laboratory, who designed the Drosophila Rosy-the-Riveter protagonist of the book and produced all of the drawn illustrations. Kristopher convinced Drosophila Workers Unite! will teach you how to work with flies in the lab, from putting me to include a section on hidden figures in the chapter on “Drosophila them to sleep to designing and setting up genetic crosses. If you read the first few researchers then and now.” I am also indebted to Jonathan DiRusso, who as chapters before your first day in lab, you will thoroughly impress your teacher! The an undergraduate in my lab developed our protocol for imaging adult flies book starts with pointers on how to find your way around a fly lab and ends with online (without shadows!) and produced the majority of photographs in this book. I resources that you can use whether you are a beginner or an expert. In addition, you would also like thank Sonia Hall for encouraging me to transform the original can learn about the five Nobel prizes awarded for fly research and pick up some facts version of this manual, which I wrote to accompany a laboratory course, into about the first fly researchers that your lab head and/or instructor probably doesn’t a full-fledged manual for all Drosophila beginners. Lastly, I am indebted to know. colleagues for their insightful comments on drafts of this manual including: Laura Quilter, Sonia Hall, Gregory Davis, Michael Dietrich, Pamela Geyer, Note to teachers: If you are teaching a unit on Drosophila, this book will help your Norbert Perrimon, and Michael Levine. students hit the ground running. It explains why Drosophila is a powerful model organism, beginning with a discussion of the Drosophila genome and available Author’ s N o t e genetic tools. This book will also help you teach students how to distinguish females from males, how to set up genetic crosses, how to recognize common This book explains the tricks of the trade of working with Drosophila. dominant markers, and how to use the Gal4-UAS gene expression system. Unique Normally these tricks are passed down from researcher to researcher, not to manuals on fly pushing, this book also discusses Nobel Prizes awarded for learned from a book. In fact, I never intended to write about the basics of Drosophila research, as well as early “hidden figures” in Drosophila research who doing fly work. However, over the last six years, this book basically wrote developed many of the tools that we use today, including the CyO balancer and S2 itself while I trained over 120 students in laboratory courses and over 80 tissue culture cells. students in my research laboratory. Drosophila Workers Unite! is intended to be a staple for all fly labs. 2 3  Table of Contents Chapter 1. Chapter 10. D ro s o phi l a : G rea t a n d M i g hty! p.6 D rosophila Researchers: T hen and Now p. 76 Chapter 2. Chapter 11. The F l y L i fe Cyc le: P ro s a n d Con s p.10 Resources for Students and Teachers p. 92 & H ow to M a i n t a i n I t Chapter 12. Chapter 3. p.20 Major D rosophila Stock Centers p. 94 Int ro d u ct i o n to CO 2 a n d Fly Pus h i n g Chapter 4. References p. 96 H ow to s e t u p f l y c ro s s es p.28 Chapter 5. D ro s o phi l a C h ro mo s o mes, G e n e ti c Symbo ls , an d p.3 8 Ge no t yp es Chapter 6. p.42 Ge ne t i c Ma rkers Chapter 7. p.5 6 D ro s o phi l a B a l a n cers Chapter 8. The G al 4 - UA S G en e E x p ress i o n Sys te m p.6 0 Chapter 9. D ro s o phi l a N o b el P ri z es p.70 4 5  1. Drosophila: Great and Mighty! It may surprise you to learn and genetic pathways that function that Drosophila researchers in humans. often forget that fruit flies are insects. Indeed, we often think of What makes Drosophila great and Drosophila as simplified humans or mighty is that it enables us to do inexpensive mice. This is because experiments that would not be on a molecular level, Drosophila feasible in humans, or most other are surprisingly similar to us! model organisms. What kinds of experiments? Experiments that we The molecular similarities between want to perform in living animals Drosophila and humans came into (“in vivo”) on a massive scale, such sharp focus when their full genome as a genetic screen to uncover all sequences were published at the the genes involved with how cells Drosophila turn of the century (Figure 1.1; communicate. Experiments where biology reflects many facets Adams et al., 2000; Lander et al., we want to turn specific genes on of human 2001; Venter et al., 2001). It turns in specific cells, at specific times, biology from the mechanics of out that Drosophila has almost and at specific levels. Experiments cell division and as many genes as we do—14,000 where we want to learn about cell polarity to developmental genes compared to our 21,000 how embryos develop, or how biology, behavior, genes. Even more impressive, over animals age, or how we learn, for and disease. The protagonist 65% of human disease-associated example. Experiments where we in this book is genes have a correlate (homolog) in want to test the limits of genetic Rosy (one of the first mutants Drosophila (Ugur et al., 2016). This engineering. discovered) and means that you can use Drosophila an homage to the inspirational to learn about many of the genes Rosie the Riveter. 6 7  In Drosophila, we can do all of the American Cancer Society. these kinds of experiments in living animals on a massive scale. Over the course of the last 100+ years, researchers have built Textbooks like to say that what genetic tools that make doing makes a model organism great genetics in Drosophila easier and is that it has a short life cycle, more powerful than any other is small, and is easy to grow in a multicellular animal on earth (with lab. These details are somewhat the exception of the roundworm C. boring and are neither necessary elegans, which is admittedly equally nor sufficient to make a model awesome). These tools include: organism great. Drosophila has all (1) visible “markers” that make it these features, but so do countless possible to track the inheritance other small animals. of any linked gene, (2) balancer Figure 1.1 chromosomes, which make it The Fly Genome What Drosophila has, that your possible to keep stocks with lethal The complete run-of-the-mill fast reproducing, mutations, and (3) the Gal4-UAS sequence of the fly genome was small and friendly animal does not system which makes it possible to published in the have, is a long history of well- express transgenes in any tissue at Journal Science in 2000. The entire funded research. Drosophila has any time in the life cycle of the fly. issue is devoted been cultured in the lab for over to understanding what the 100 years with decades of support sequence means from the Carnegie Institute, the in relation to the development, National Institutes of Health (NIH), physiology, the National Science Foundation evolution, and similarity to (NSF), the Howard Hughes humans, of Medical Institute (HHMI), as well Drosophila. as countless smaller foundations including the March of Dimes, the American Heart Association, and 8 9  2. The Fly Life Cycle: Pros and Rosy’s Origin Story Cons & How to Maintain It egg There are admittedly some to new vials or bottles to keep drawbacks to working with them from overpopulating and Drosophila. First, they are alive. destroying themselves within the parents Second, they like to mate even confines of their homes. Every when you don’t want them to. Drosophila researcher occasionally Third, they cannot be stored forgets to transfer flies in a L2 cryogenically. What this means is timely fashion, resulting in an L1 that you have to take care of them. “overpopulation catastrophe”. If you experience this, the good Adult flies have one major mission news is you’ll be very careful in in life, which is to make as many the future to transfer your flies offspring as time permits. At frequently! the standard so-called “room temperature” of 25°C, adult flies develop from fertilized eggs in To p i cs covered i n t hi s Wa n d e r i n g L 3 9–12 days. This is great because it Chap ter : means you can get lots of flies fast. But it also means that you have 1. Fly Life Stages to stay on your toes, keeping flies 2. Fly Life Cycle happy and making sure they don’t 3. Standard Temperatures for get overcrowded. Rearing Flies: 18°C, 25°C, 29°C 4. How to Keep Your Own Stock pupae To compensate for the fact that Collection flies are constantly reproducing, 5. Quick guide to flipping flies Rosy! you will have to transfer flies 10 11  F ly Li fe S t a ge s Fly eggs can be easily collected into a multicellular animal: Dr. Fly Life Cycle Although Drosophila researchers by the hundreds to thousands to Christiane Nüsslein-Volhard, Dr. Regardless of which stage of the often see the fly as a simplified study embryogenesis. In fact, the Eric Wieschaus, and Dr. Edward fly you work on, you will need to human, it is indeed much more 1995 Nobel Prize in Physiology Lewis. If you are interested in cell have an idea of the complete fly than that. With flies you can or Medicine was awarded to and organismal growth, larvae are life cycle to plan your experiments. study 4 completely different life three Drosophila researchers an excellent choice to study, as forms, depending on the stage you for their work using Drosophila they undergo tremendous growth If you want to work with eggs decide to study: embryos, larvae, embryos to dissect the genetics in just a few days. On the other and embryos, plan on doing pupae, and adults (Figure 2.1). of how a fertilized egg develops hand, if metamorphosis is your experiments within minutes to favorite subject, you can study hours after eggs are laid by their pupae which undergo radical mothers. If you want to work with metamorphosis involving massive larvae (also called instars), you cell death and the growth of new have a choice of three stages, body parts from specialized cells, called L1, L2, and L3. You can housed in 19 structures called collect L1 larvae about 24 hours imaginal discs. Adult flies are also after eggs are laid. However, L1 a popular choice to study, as their and L2 larvae are very small and molecular and cellular biology, can be difficult to study. L3 larvae physiology, and behavior provide are the most frequently used in Egg Larva insights into human biological experimental studies: they are the processes and diseases. For largest and most robust. With a example, the discovery of innate pair of #5 forceps, you can easily immunity in humans owes it transfer L3 larvae from vial to vial, origins to research in Drosophila. and with some practice, you can The scientist who discovered the dissect imaginal discs out of them. molecular basis of innate immunity Expect L3 larvae to crawl out of Figure 2.1 in Drosophila, Dr. Jules Hoffman, the food about 5 days after eggs Four Stages of was awarded the 2011 Nobel Prize are laid. Once an L3 crawls out of Drosophila: Egg, in Physiology or Medicine. the food, it will crawl as a larva for Larvae, Pupa, Pupa Adult Adult 12-16 hours and then pupate. 12 13  During the first 12 hours of Standard Te m pe ratu re s for Re aring Flies pupation the pupa is called a “pre- The rate of fly development from egg to adult is described in this manual pupa” and can be distinguished for the standard temperature of 25°C, often called “room temperature.” You from the other pupal stages can speed up fly development by raising progeny at 29°C, where it takes 1 because it is translucent white. only 7-9 days for a fertilized egg to develop into an adult. However, flies Pupae darken as they age, are generally less healthy at 29°C, so do this only if you need to. You can transitioning from white, to also slow down fly development by raising flies at cooler temperatures. For 2 3 caramel, to dark brown. You can example, if you raise flies at 18°C, it typically takes two weeks for a fertilized gauge when a pupa is about to egg to develop into an adult. 18°C is ideal for keeping stocks because it hatch based on how dark it is. means that you can flip the vials less often than at 25°C. Most fly labs have incubators dedicated to each of these standard temperatures (Figure 2.3). In Figure 2.2 you can see pupae at different stages, including dark pupae (e.g., #2), which will hatch in the next 24 hours. About a day before a pupa hatches, you can Figure 2.2 see adult structures through its The Pupae (pyoo-pay) This funny sounding word comes from latin pupal case: the wings appear as “doll”, not because pupae are two black structures running along 18 particularly cute, but because they are immobile. Don’t let their the length of the pupal case, and immobility fool you though! Pupae the eyes appear as two prominent can tell you a lot about your fly culture. If most are young spots in the head region. When a (e.g., pupa #1), you can look forward fly hatches out of the pupal case to newly hatched flies in about 5 18° 25° 29° days. If most have visible structures (referred to as eclosion), it leaves like wings and pigmented eyes behind its empty pupal case, which Figure 2.3 (e.g., pupa #2), you will be able to collect virgin flies within the next you can spot by looking closely at Fly incubators 24 hours. If most of the cases are the walls of old vials or bottles. In are standard empty (e.g., pupa #3), that means lab equipment the culture is old and should be total, a pupa requires about 5 days and typically set flipped to a new vial or bottle soon. to develop and hatch (eclose) into at 18°C, 25°C, or 29°C. an adult fly. 14 15  H ow to Ke e p Yo u r O w n S to ck Co llect i o n Gu ide to flipping flie s Most fly labs keep a fly “stock collection” which consists of two vials (copies) of every fly stock used in the lab. The flies in the stock collection are typically not used in experiments directly. Instead, they are simply cotton cotton 1 maintained and available when a researcher needs a copy of a stock. In most labs, the stock collection is maintained by a designated individual with lots of experience raising flies. If you want a stock from the collection, this person old new will flip the flies from the stock collection to a new vial and give it to you to vial vial keep for your experiments. This method of handing out copies of stocks helps to ensure that the stocks in the collection stay pure. The key to keeping any stock collection is to flip the flies frequently so that 2 4 they do not get overcrowded. This means that you should transfer your stocks to new vials on a regular schedule. Most labs maintain their stocks 3 new old at cool temperatures in the range of 16°C-18°C, because flies grow more vial flip together vial slowly at these cooler temperatures, thereby giving you several weeks before having to transfer them to new vials. If you are lucky enough to have upside down! access to an 18°C incubator, you can keep your stocks healthy by flipping old new them to new vials on a monthly schedule. However, if you keep your stocks vial vial at room temperature or 25°C, you will need to transfer your stocks to new Figure 2.4 vials every two weeks. How to flip flies. Before flipping your first stock 5 of cherished flies, you should practice with a vial of flies from the old new vial vial trash. Review the steps to the left and ask an experienced fly pusher to guide you. 16 17  S toc k M a i n te n a n ce Keep each stock in two separate vials, side by side in an 18°C incubator as follows: Day 1: Flip your flies to a new vial, Tip: 18°C is the preferred Vial #1 Vial 1 Vial 2 Figure 2.5 temperature for maintaining your stocks of flies because at Day 2: About 16-24 hours later, Maintaining Fly Stocks You should flip flies from Vial #1 to a new vial, plan on flipping Vial #2. your 18°C fly stocks from the 18°C they develop slower and designated Vial #1 to a new Vial #1 every 4 weeks. If you follow this method, you will have your stock in two vials. Vial #1 looks live longer! empty, but it is not! It has all the fertilized eggs that were laid in the 16-24 hour window that the adults were present in the vial. Vial #2 has the parents of the fertilized eggs that are in Vial #1. See Figure 2.5. After adults have lived in Vial #2 for an entire month, the vial will likely be overcrowded with adult flies and the food will likely appear “soupy,” which is a side-effect of having too many larvae churning the food. If you look closely at the adults they will likely have battered wings as a result of age and overcrowding. You may wonder then, what the point is of keeping Vial #2 around? It is simply a backup vial. For example, Vial #2 can come in handy when you want adults before they have hatched from Vial #1. 18 19  3. Introduction to CO 2 & Fly Pushing Every lab that works with flies Note: It is essential to ask must have a way to temporarily someone for help before working put flies to sleep so that with your lab’s CO₂ tank and researchers can examine them, regulator. The pressure inside count them, and set them up in a CO₂ tank is enormous and genetic crosses. These skills are dangerous if not handled correctly. collectively called “fly pushing.” Again, do not work with the CO₂ tank until trained by someone in There are many ways to put your lab! flies to sleep. In the olden days, researchers used ether vapors to put flies to sleep. However, this practice has largely been replaced To p i cs covered i n t hi s with safer methods that include Chap ter : ice, FlyNap (triethylamine), and carbon dioxide gas (CO₂). 1. CO₂ tanks, needles, and flypads 2. Getting the right flow of CO₂ CO₂ is the most commonly used 3. Getting CO₂ into fly vials method in research labs because it 5. What if flies start to wake up? is highly effective and easy to work with. This manual focuses on how to use CO₂. 20 21  CO₂ t a n k s , n e e d le s , a n d f l ypads Figure 3.2 A full tank of CO₂ has a PSI (pounds per square inch) of 750. You may have CO2 Regulator heard that if a tank were to fall over without a safety cap it would shoot Tank regulators typically through the wall like a torpedo. This is not a myth! For a real-live view, see have two gauges: one shows the pressure in demonstration by MythBusters. To avoid catastrophes make sure your tank the tank and the other is fully secured, as shown in Figure 31. shows the pressure flowing from the tank to the fly stations. The gauge on the right shows tank pressure is 500 PSI and the guage on the left shows pressure flowing to the fly stations is 10 PSI. To regulate the amount of gas released from the CO₂ tank, three control devices are used: a regulator that enables gas to flow from the tank itself, a quarter turn value that enables gas to flow at your fly work station, and a clamp to control to flow of CO₂ through the needle at your fly work station. Figure 3.1 Typically, the tank regulator (Figure Working Under 3.2) is set to release CO₂ at about Pressure CO₂ 10 pounds per square inch (PSI), tanks are under enormous Once the gas is turned on at the pressure and regulator, you can control the flow must be secured with a belt (as of CO₂ with the valve at your fly shown) or a boot. station. 22 23  Figure 3.4 Get t i n g t h e r i g h t f low to estimate the right amount of of CO ₂ at yo u r s t a t i o n pressure. Of course, the final test Microscope & Fly Station Fly Most commonly, you will have is how the flies behave. If they stations deliver Fly work station showing CO2 to the fly pad a “quarter-turn” valve at your start to wake up during your work, microscope, light source, fly pad, and a needle. The microscope station (Figure 3.3). you probably need to turn the fly pad is used paint brush, and fly morgue for sorting flies Do not open it all the way valve a little further open. and the needle is because it will likely allow too used to put flies to sleep in vials much pressure locally and will CO₂ at your fly station typically (as shown) and burst apart the tubing. As the and has two outlets: (1) a fly pad bottles. tubing bursts, it may knock over and (2) a needle (Figure 3.4). the morgue or other glassware leading to an unpleasant start to Use the needle to put the flies Remember to hold the vial upside your day in the fly room. in vials and bottles. As shown in down when putting flies to sleep. Figure 3.4, hold the vial or bottle This is to ensure that they fall You should turn the valve slowly upside down while gassing the asleep on the cotton, not the food! until you have “just enough” flies with CO₂ so that the flies fall CO₂ flowing from the needle. asleep on the cotton rather than Get someone in your lab to in the food. Once you put flies demonstrate what “just enough” to sleep in a vial or bottle, you CO₂ is for your system. Typically can gently tap them onto the fly people use the sound of CO₂ pad. The fly pad is engineered to flowing from the needle, or the distribute flowing CO₂ to the flies, Paintbrushes are the most feel CO₂ flowing from the needle thereby keeping the flies asleep. commonly used tool to move flies around on the fly pad. Some people prefer to use feathers or Figure 3.3 even forceps. Whichever you Quarter Turn Valve When the valve handle is perpendicular to you, it is fully closed, and use, remember your job is to be a when it is pointing at you, it is fully open. “gentle giant.” Typically, it’s a bad idea to open the valve all the way. Turn the handle slowly and stop once you have a “good flow” of CO2 from the needle. 24 25  Once you have flies on the fly pad, you do not need to keep CO₂ flowing What if flie s star t to wake u p? through the needle. If a clamp is available, use it to stop the flow of CO₂ to First, do not panic. Depending on how awake they are, there are different the needle once the flies are on the pad. steps you can take. Hopefully you are calmly reading this in advance of any wake-up catastrophes. You can safely keep flies on the pad with CO₂ for 10 minutes. Don’t put so many flies on the pad that it takes you more than 10 minutes to sort them! What if flie s star t to twitch! If you keep flies on the pad for more than 10 minutes, they may become If flies start twitching on your pad, make sure that CO₂ is not still flowing to sterile, which is no fun when you’re doing fly genetics. your needle. If it is, clamp it shut to direct more CO₂ to your flypad. Also, cup your hands over the flies to concentrate the CO₂ around them. If this doesn’t Get t i n g CO ₂ i n to v i a l s help, check that the tank still has gas! First open the CO₂ valve and adjust until you have a “good flow” of CO₂ from the needle. Then pick up the vial or bottle of flies and turn it upside What if flie s star t walk ing! down in your hand. Next, insert the CO₂ needle between the cotton plug and If flies start to walk, you’re in trouble. As fast as possible, brush them into a sidewall of the vial/bottle. Keep holding the vial/bottle upside down as the vial! If they are walking off the pad, you must kill them. See Massive Wake flies fall asleep onto the cotton plug. You can help them fall down faster by Up Catastrophe! gently tapping the sides of the vial/bottle. Once it looks like all the flies are sleeping, wait an extra 5 seconds before pulling out the needle, to ensure Massive Wake U p Catastrophe that the flies are deep asleep when you transfer them to the fly pad. If flies are starting to stream off your fly pad, there’s no other solution than to kill them all. Ask for help! But if no one’s around, you’ll have to do Keep the vial/bottle upside down throughout the process. Once they are it yourself! Your choices are to crush them by hand (with or without paper asleep, gently remove the cotton plug and tap the flies onto the fly pad. towels) or to drown them with a handy squirt bottle of water or ethanol. If Then put the cotton back in the vial/bottle. We recommend that you you use the drowning method, you will have to follow-up with paper towels keep the vial/bottle on its side so that any remaining flies have a chance to clean them off the bench top. If you use the crushing method you will to wake up without getting stuck in the food at the bottom of the vial/ have to follow up with ethanol, to clean up the mess. Don’t forget to wash bottle. Remember, do not put more flies on the pad than you can sort in 10 your hands after! minutes! 26 27  4. How to Set Up Fly Crosses Setting up fly crosses is the bread- To p i cs covered i n t hi s and-butter of being a fly geneticist. Chap ter : As long as you know how to identify females of one genetic 1. Sexing flies stock and males of another, you 2. The importance of virgin females can combine them in a vial and 3. Collecting virgins get fertilized eggs. This process is 4. The clock method called “crossing flies.” 5. Clearing vials and bottles 6. Visual virgins To cross flies, you will need to 7. Collecting virgins using a genetic know how to identify females and trick males. The process of identifying 8. Specifics of using heat-shock the sex of flies is called “sexing inducible Hid flies.” In this chapter we will teach 9. Setting up crosses you how to sex flies and set them up in fly crosses. 28 29  Female fly S ex i n g f li e s The im por tance of v irgin fe m ale s Sexing flies means telling the Fly genetics has been successful over the last 100 years because flies are difference between females easy to cross and their progeny have many phenotypes you can track. But and males. It’s easy. Turn them before you begin, you need to know something about fly sex that your on their back and look at their parents may not have told you: female flies like to control the fertilization of genitals (Figure 4.1). Female their eggs by storing sperm in an organ called the spermatheca. Each time a genitalia are pale in comparison to female mates, she stores the sperm in her spermatheca which she then uses male genitalia. Males can also be to fertilize her eggs. This means that for you to set up meaningful crosses, identified by dark bristles, called you need to use virgin females. Using virgin females is the only way to sex combs, on their front pair of Male fly ensure that the offspring from the crosses you set up are from your crosses legs. It’s challenging to spot the and not from earlier matings that the females set up themselves. sex combs but gratifying once you do. So, give it a try! However, don’t rely on sex combs to identify male Colle cting v irgins flies because it takes too long. There are three ways to collect virgin females. Most researchers use the Instead, get used to looking at fly “clock method,” which is based on the fact that females cannot mate for genitalia to identify females and several hours after they hatch (eclose) from the pupal case. The clock males. method enables you to collect large numbers of virgin females, but takes careful planning. Another commonly used method is the “visual method,” which enables you to collect the youngest of the virgin females based on Male sex combs their appearance, which for the first hour after hatching is visually distinct from typical adult flies. This method is a convenient way to collect virgins without any planning on your part. But it is not a good method to use when Figure 4.1 you need a lot of virgins because “visual virgins” represent only the youngest Sexing flies of all the virgin flies in your vial. The third method requires that you use special fly stocks that kill off all the males before they have a chance to mate Simply turn flies on their back to check with the females—in these stocks, all the females remain virgin because whether they have there are no males for them to mate with. female or male genitalia. Look for sex combs as a last resort! 30 31  T h e cloc k m e t h o d into lab every 8 hours to collect Cle aring v ials and bottle s Luckily for us, when female flies virgin females. Luckily, you can For the timing method to work, it is essential that you master the art of hatch at 25°C, they cannot mate avoid this simply by having your “clearing” vials. Clearing is a polite word for killing all the adults. The most for the first 8 hours of their adult flies hatch at 18°C rather than ethical way to do this is to put the flies to sleep with CO2 before dumping life. So, if you collect female flies in 25°C. This is because at 18°C them into the morgue. After dumping out the sleeping adults, look carefully the first 8 hours of their adult life, fly development slows down, in the vial/bottle and along the walls, to make sure there are no lingering they will not have mated and you stretching the window that newly adults remaining. If there are, tap them out, or use a disposable wooden set them up in crosses that you eclosed females remain virgin dowel to push them into the food (which is another way of killing them). completely control. from 8 hours to 18 hours. This means that you can “clear” a vial How do you know if a fly is 8 hours or bottle of all the adults, and Visu al virgins old or younger? The trick is simply when you come back the next day, When flies hatch, they have a distinct “newly hatched” white puffy to use “clearing” and timing: if you within 18 hours, all the new adult appearance for their first hour of life that makes them easy to spot. Females clear a vial or bottle of all adults females will be virgins. This is great with a newly hatched appearance must therefore be virgins. However, males in the morning and then return 8 because it means you can collect that just hatched also have a newly hatched appearance, so be sure to look hours later in the afternoon, any lots of virgins and still have dinner carefully at the genitals of newly hatched flies so that you select only the adults in the vial/bottle must be 8 and a full night of sleep. Typically, females when collecting virgins. hours old or younger. researchers clear vials and bottles at 4 PM and collect virgins the Another visual marker that distinguishes young flies is a dark abdominal spot, If you think about it, the clock next day before 10 AM (Figure Figure 4.2 which is typically present in flies for their first 2-3 hours after hatching. The method could have you running 4.2). The Clock dark spot is the fly equivalent of meconium, sometimes called flyconium, Method of and is the remnants of the fly larval gut inside the intestine of the adult. Collecting Virgins If you use this Flyconium is typically expelled within the first 3 hours of an adult fly’s life. If method you will you forgot to clear your vial or bottle, you can still collect the youngest of the likely keep the vials/bottles at virgins because of their distinct appearance. 18 degrees and collect flies on an 18 hour schedule, making sure the vials are clear of all adults at 10 PM AM AM and 6 PM. 32 33  Collect i n g v i r g i n s stock, the other to subject to heat- Figure 4.3 u si n g a ge n e t i c t r i c k shock to collect virgin females. There is an easy genetic method Heat shocking in a water bath to get virgin females that doesn’t The stocks for this purpose have a Heat-shock involve running around 8-hour and experiments are special Y chromosome engineered most commonly 18-hour clocks to collect them. with a lethal transgene, called done by The method is to use a stock with submerging fly “heat-shock inducible Hid.” The vials and bottles male flies that are genetically Hid transgene encodes a protein in a 37°C water engineered to die before they bath. Be sure to that activates programmed cell keep the cotton hatch. This kind of stock only death (apoptosis). It is engineered tops above the gives rise to females, which in the water though! under the control of a heat-shock absence of their male brothers, promoter which activates gene remain virgins indefinitely. This expression when flies are placed at allows you to collect virgins at your 37°C (see Figure 4.3). Since males leisure to set them up in genetic are the only flies in the stock with crosses. a Y chromosome, when you place flies at 37°C, only the males die. If you think about it, a stock of flies Typically, the heat-shock is carried must have both females and males out in a vial of fly embryos, aged to make new generation after 0 to 24 hours old. The heat-shock generation of progeny. So how can causes all the male embryos to a stock persist if all the males are die while all the female embryos engineered to die? The trick is that continue developing, eventually they are engineered to die only under becoming larvae, then pupae, and certain conditions, such as a “heat- finally adults. Since there are no shock” in which the fly vial/bottle is males after the heat shock, the placed at 37°C for 1-2 hours. You hatched females remain virgin should have at least two copies of indefinitely. You can get Hid fly the stock: one to keep at normal stocks from the Bloomington temperatures to maintain the Drosophila Stock Center. 34 35  S p eci f i cs o f u s i n g h e a t- s ho ck i nduci b le H i d Se tting u p crosse s If you forget to flip your cross to The Hid transgene works best when it is expressed during the first 0-24 Once you know how to put flies new vials, the food will become hours of the fly’s life, while it is still an embryo. To use this method, you to sleep, and how to collect virgin oversaturated with larvae. Under need to get a collection of 0-24 hour fly embryos. Sounds hard, but it’s females, it’s easy to set up fly these conditions, the larvae will easier than it sounds! Simply put adult flies in a vial/bottle of freshly made crosses. You simply count out not get sufficient food, they will fly food overnight. Label the vial/bottle “embryo collection.” When you about 5-8 sleeping virgin females develop poorly, and they will often come back the next day, flip the adults to a new vial/bottle. The vial/bottle and 5-8 sleeping males and put be undersized or “minute.” labeled “embryo collection” now contains a collection of embryos that are them in a new vial where they 0-24 hours old. Put this vial/bottle in a 37°C incubator (preferably half- will wake up, mate, and lay eggs. To figure out how many times to way submerged in a water bath) for 1-2 hours to ctivate expression of the It is best to keep vials of sleeping flip your cross, think about what Hid transgene (Figure 4.3). Then put the vial/bottle at 25°C to enable to flies on their sides; once the flies fraction of the progeny will be surviving females to develop. In about 10 days the adult flies—all female— wake up you can right the vial. The the genotype that you want and will hatch. In rare cases you may see a few adult males too. These are called mating flies are referred to as a how many flies you need with that “escapers” because they escaped death. Luckily, male escapers tend to be “cross.” genotype. On average, expect sterile, so your females will very likely still be virgins. about 100 offspring from each vial When female and male flies of a healthy cross with 8 fertile are first introduced, they need females crossed to 8 fertile males. a couple of days to become acquainted with each other. After Once you calculate how many that, the females will start laying progeny you need to collect, check lots of fertilized eggs every day. with your mentor to make sure you Therefore, it’s a good idea to flip have a good plan for raising and your cross to new vials every flipping the cross. one to two days after the initial “getting to know you” period. Note: Use young flies and fresh Flipping the cross to new vials food to increase the number of every one to two days will ensure progeny from your crosses. that the vials do not become overcrowded with progeny. 36 37  5. Drosophila Chromosomes, Genetic Symbols, & Genotypes Drosophila, like us, is a diploid organism. This means that it has two copies of each chromosome set: it gets a complete set of chromosomes from mom and a complete set of chromosomes from dad. In humans, a complete set (1n) consists of 23 chromosomes. Drosophila is simpler: a complete set (1n) consists of 4 chromosomes. The first pair are the sex chromosomes X and Y, and the other three pairs are autosomes, designated chromosome 2, 3, and 4. Top i c s cove re d i n t h i s C h ap ter : 1. The 4 chromosomes: 1 pair of sex chromosome 2. Sex chromosomes 3. Writing genotypes 38 39  Chr. 1 (X) IIIIIIIIIIIIIIIIIII Drosophila Ge notype s IIIIIIIIIIIIIIIIIII A fly that is homozygous for a Chr. 2 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Genotypes are written for each of loss of function mutation in the the three major chromosomes: white (w) gene, which is on the X Chr. 3 1, 2, and 3. The 4th chromosome chromosome is written: I and the Y chromosome have so w-/w- ; +/+ ; +/+ Chr. 4 101 102 few genes, that they are usually ignored for the routine work of Since this above genotype is Chr. Y most fly geneticists. homoygous, it can be abbreviated: w- ; + ; + Figure 5.1 Drosophila chromosomes. Flies, like us, are diploid. They have The X chromosome is designated as Chromosome 1. Each major chromosome “arm” is subdivided into twenty two copies (homologs) of every Fly geneticists often skip writing cytological units based on Calvin Bridges labeling system, as shown by the numbers within the chromosome arms. chromosome. The genotype of minus signs because they typically the homologs is separated by a only highly mutant alleles. Thus, slash mark “/” and genotypes of it is more common to write the T h e 4 ch ro m o s o m e s o f D ro so p hi la and Y different chromosome types are genotype of a homozygous white separated with a semicolon. mutant as: The Drosophila genome consists of 4 pairs of chromosomes: w; +; + Chromosome 1 (X) = About 20% of the genome Females have the chromosomes: Chromosome 2 = About 40% of the genome 1/1 ; 2/2 ; 3/3 A fly that is heterozygous for a Chromosome 3 = About 40% of the genome mutation in white, is written: Chromosome 4 = dimpled/narrow eyes can be detected earlier in the life Curly —> curly wings cycle of the fly, such as “Tubby,” Stubble —> short bristles which results in short tubby pupae. Tubby —> short tubby pupae 42 43  The “ white plu s” ( w+) m arke r Figure 6.1 w+ is the most commonly used marker in Drosophila. It is dominant and produces red eye color. (Figure 6.2) Most fly labs have the “Learning to Fly” poster How to de te ct the w+ m arke r on display. It’s a fantastic resource Any student can detect the w+ marker because eyes are among the most for identifying the prominent features of fruit flies. You don’t need a dissecting microscope: you most commonly used markers. can see their large red eyes by looking at them through the vial, or on a piece Reprinted with of fruit when you find flies in your kitchen. permission from Richard Behringer and Georg Halder. The white ge ne nom e nclatu re : w vs. w+ v s. w- w is the genetic abbreviation for the gene “white”. The wild type version (allele) of white is denoted “w+” and it results in flies with red eyes. The mutant version of white is denoted “w-”. Flies that have only the w- version of the white gene have white eyes. Figure 6.2 Close up of fly eyes: red and w+/w+ w-/w- white 44 45  H ow w h i te go t i t s n a m e Figure 6.3 You may wonder why the gene that normally gives flies red eyes is called white. It The wild type, turns out that most genes are named for their mutant phenotype! functioning copy of white, called w+, became a powerful The white gene encodes information to make a transmembrane pump that marker because it transports pigment precursors that are required to make the red pigment you is so easy to detect. Just as it was simple normally see in fly eyes. If a fly lacks a functioning copy of the white gene, it will for Morgan to spot a be unable to transport pigment precursors and as a result the fly will have white white eyed fly among hundreds of red eyes. On the other hand, if a fly has even just one functioning copy of the white eyed flies, as shown gene, it will be able to transport pigment precursors, resulting in an eye that is here, any student can easily detect a red (or some shade of red as described below). red eyed fly among hundreds of white eyed flies. Discovery of the white gene led to many insights and a Nobel Prize in 1933. The white mutant was discovered by Thomas Hunt Morgan who was a renowned professor of developmental biology at Columbia University. He had been breeding flies in hundreds of glass milk bottles hoping to discover a spontaneous The discovery of the white mutant (w-), led Morgan and his mutant or “sport.” After two years of searching unsuccessfully for a single students to make several insights into the relationship between mutant fly he was ready to give up (Kohler, 1994). But one lucky day in 1910, genes and chromosomes. In 1933, Morgan was awarded the he spotted a fly with white eyes among hundreds of flies with red eyes (Figure Nobel Prize for these insights, which all began with the white 6.3). He named this fly “white” because it had white eyes. It was one of the first mutant. Morgan shared his Nobel prize money with his two top Drosophila mutants ever discovered and saved. students—Alfred Sturtevant and Calvin Bridges—both of whom began in his lab as undergraduates and propelled much of the Morgan isolated the white eyed fly, a male, and crossed it to normal (wild type) research leading to the Nobel Prize. red eyed females. All of the F1 progeny had red eyes, indicating that white was recessive to red. When Morgan crossed the F1 red eyed progeny with each The mutation in white was mapped and cloned several decades other, he found that ¼ of the F2 offspring had white eyes, confirming that the later in the 1980s (O’Hare et al., 1984) and found to reside in white mutant was due to a recessive mutation. However, this was not a simple a gene that encodes a transmembrane pump, whose molecular recessive mutation because all of the white eyed F2 flies were males (Morgan, function is to transport small molecules, including pigment 1910). This observation, plus results from additional crosses, led to the discovery precursors, across membranes (Mount, 1987). of X chromosome-linkage and proof that genes are located on chromosomes. 46 47  white (w) is at position 1-1.5 Figure 6.4 w+ and transge nic flie s Chr. 1 The endogenous X white gene is The w+ marker is most commonly used marker to detect and track located on the transgenic flies. Scientists make transgenic flies by engineering X chromosome at cytological stretches of DNA in the lab and inserting the engineered DNA into the position 1-1.5. fly genome. The engineered DNA typically encodes two linked genes: the gene being studied (which is rarely possible to detect on its own) and a wild type copy of the white gene (w+). The engineered DNA is T h e gen et i c s o f t h e w h i te gene at i t s no r mal then inserted into a fly genome that lacks a normal copy of the white ( en dogeno u s ) lo c u s gene at its endogenous locus. Without the engineered DNA, this The endogenous white gene is located on the X-chromosome and has the genome instructs the development of flies with white eyes because genetic symbol “w” (Figure 6.4). it lacks a functioning copy of white. However, a fly that inherits the engineered DNA will get a copy of w+ and therefore will have red A Drosophila female has two X chromosomes typically has one of the eyes. In this instance, w+ serves as a marker gene that lets scientists following genotypes and corresponding phenotypes: know that the fly is carrying transgenic DNA. w-/w- = white eyes w+/w+ = red eyes This is a powerful trick that lets researchers track the inheritance of w+/w- = paler red eyes *this is because the degree of pigmentation is any engineered genes they wish to study: regardless of how hard it is sensitive to the dosage of the w+ gene product. to detect the engineered genes, by linking the engineered genes to w+, they can follow the genes through generations because the linked A Drosophila male has one X chromosome typically has one of the following w+ transgene will give flies red eyes. genotypes: w-/Y = white eyes The ge ne tics of {w+} in a w-/w- back groun d w+/Y = red eyes *these males will have red eyes that are equal in w+ is a useful marker to track transgenes as long as it is inserted into intensity to females that are w+/w+ due to a phenomenon called dosage a genome that lacks a wild type copy of white. Flies that lack w+ at compensation! the endogenous locus have the genotypes w-/w- (female) and w-/Y (male), and are normally white eyed. Since since w+ is dominant to w-, flies that have a w+ linked transgene can be distinguished by their red eye color. The w+ transgene is often denoted with brackets, {w+}, to distinguish it from the endogenous gene. 48 49  S h ades of re d gene expression as the two X chromosomes in females. As a result, males Interestingly, w+ is a “dosage with the hemizygous w+ genotype, w+/Y, have eyes that are as dark red as sensitive gene”. The intensity of homozygous w+/w+ females! red pigment produced by the transport activity of the w+ gene The eye color that results from {w+} transgenes ranges from pale yellow depends on how much w+ protein to dark red. This is because the amount of gene expression of any gene, is produced. This means that flies including transgenes, depends on their location in the genome. Transgenes with two copies of the w+ gene that land in highly compacted regions called heterochromatin, tend to be typically have darker red eyes than poorly expressed. As a result, very little white protein is produced, resulting a fly with only one copy of w+ in eyes that are pale yellow to light red. Conversely, transgenes that land (Figure 6.5) in open regions of the genome called euchromatin, are highly accessible to transcriptional machinery, resulting in high levels of white mRNA production. For example, females with a As a result, high levels of white protein are produced, resulting in eyes that homozygous w+/w+ genotype are intensely red. have darker red eyes than females with a w+/w- heterozygous Su m m ar y on the u se of the w+ m arke r genotype. Heterozygous w+/w- Most flies in a modern genetics labs are transgenic. They lack endogenous females often have orange colored wild type copies of the white gene, but have red eyes because they carry eyes as shown in Figure 6.5. a {w+} marked transgene. In these flies, w+ is simply a marker that lets researchers know the fly is carrying other transgenic DNA of interest. Male flies have only one copy of the X chromosome and therefore can have only one copy of w+ under normal non-transgenic circumstances. However, due Figure 6.5 to a process called dosage Fly eye color compensation, genes on the can range from single X chromosome in males dark brick red— high expression of get expressed at double the rate, w+, to white—no resulting in the same output of expression of w+). 50 51  Bar (B1) is named for its mutant Wil d Ty p e E y e l d Ty p e W i n g Wi phenotype, which results in flies with Curly (Cy ) is named for its 1 mutant phenotype, which results in flies narrow eyes (Tice, 1914), as shown in with wings that curl upwards, as shown Figure 6.6. Bar is abbreviated with a in Figure 6.7. Curly was discovered by capital “B” to signify that the first mutant Lenore Ward, who in 1923 published the discovered in the Bar gene was dominant. observation that the Cy1 mutation causes (The white gene, on the other hand was two phenotypes: in one copy it causes discovered by a recessive mutation and the wings to curl upwards and in two hence the abbreviation for the white copies it causes flies to die before they gene, “w,” is lowercase). Not all alleles reach adulthood (Ward, 1923). Hence, of the Bar gene result in dominant Cy1 causes a dominant wing phenotype phenotypes. However, the allele you and is also a “recessive lethal.” Ward are likely to encounter, B1, results in a mapped Cy1 to the second chromosome Bar Eye ly Wing dose-dependent dominant phenotype as and recognized that this could be a Cur follows that differ in females and males powerful marker for mapping genes to because it is X-linked. the second chromosome. In females Curly is one of the most widely used B1/B+ = Subtle kidney shaped eyes markers in Drosophila. It is used to B1/B1 = Narrow shaped eyes mark a special version of the second chromosome called the CyO (pronounced In males “Curly-O”) balancer. You can read more B1/Y = Narrow shaped eyes about this balancer in the next chapter. Figure 6.6 Figure 6.7 The B allele is used to distinguish a special version of the X chromosome 1 Wild type vs. Wild type vs. called the FM7 balancer, discussed in the next chapter. Bar eye Curly wing. 52 53  Stubble (Sb ) 1 was Wi ld Ty p e B r i s t le s Tubby (Tb1) was discovered Wi l d Ty p e P u p a by Charlotte Auerbach (Auerbach, 1943), discovered by Calvin Bridges in 1923 and who is widely credited for establishing is named for its thick short bristles on that chemicals, such as mustard gas, the back of the fly thorax (Bridges and can be as powerful as X-rays in creating Morgan, 1923), as shown in Figure 6.8. heritable mutations (Beale, 1993). The Sb1 dominant bristle phenotype The Tb1 mutant is dominant and easy to is hard to see without a dissecting spot in L3 larvae and pupae: it is short microscope, but with proper lighting and and “tubby” compared with wild type magnification, it is easy to spot. counterparts, which by comparison are long and lean, as shown in Figure 6.9. Sb1 is used to track inheritance of Although Tb1 mutants are dominant mutations on the third chromosome. It ble Bristl by Pupa ub es during the L3 and pupal stages, they Tu b is also commonly used as a marker for St are undetectable at the adult stage. a special version of chromosome three, Therefore, if you are using this marker, called the TM3 balancer. You can read you must plan to screen for it before Tb1 more about this balancer in the next flies hatch into adults. chapter. The Tubby allele Tb1 causes a dominant visible phenotype in the size and shape of larvae and pupae and is also a recessive lethal. Tb1 is most often used as a marker for special versions of chromosome 3 called TM6, as described in the next