Drosophila Workers Unite! A laboratory manual for working with Drosophila By Michele Markstein 1 2 By Michele Markstein By Michele Markstein 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. 3 1 Acknowledgements 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, Michael Dietrich, Pamela Geyer, Norbert Perrimon, Note to teachers: If you are teaching a unit on Drosophila, this book will help your 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 Note genetic tools. This book will also help you teach students how to distinguish This book explains the tricks of the trade of working with Drosophila. females from males, how to set up genetic crosses, how to recognize common Normally these tricks are passed down from researcher to researcher, not dominant markers, and how to use the Gal4-UAS gene expression system. Unique learned from a book. In fact, I never intended to write about the basics of to manuals on fly pushing, this book also discusses Nobel Prizes awarded for doing fly work. However, over the last six years, this book basically wrote Drosophila research, as well as early “hidden figures” in Drosophila research who itself while I trained over 120 students in laboratory courses and over 80 developed many of the tools that we use today, including the CyO balancer and S2 students in my research laboratory. Drosophila Workers Unite! is intended to tissue culture cells. be a staple for all fly labs. 2 3 Table of Contents Chapter 1. Chapter 10. Drosophila: Great and Mighty! p.6 Drosophila Researchers: Then and Now p.76 Chapter 2. Chapter 11. The Fly Life Cycle: Pros and Cons p.10 Resources for Students and Teachers p.92 & How to Maintain It Chapter 12. Chapter 3. p.20 Major Drosophila Stock Centers p. 94 Introduction to CO2 and Fly Pushing Chapter 4. References p.96 How to set up fly crosses p.28 Chapter 5. Drosophila Chromosomes, Genetic Symbols, and p.38 Genotypes Chapter 6. p.42 Genetic Markers Chapter 7. p.56 Drosophila Balancers Chapter 8. The Gal4-UAS Gene Expression System p.60 Chapter 9. Drosophila Nobel Prizes 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 Adams et al., 2000; Lander et al., we want to turn specific genes on many facets of human 2001; Venter et al., 2001). It turns in specific cells, at specific times, biology from the out that Drosophila has almost and at specific levels. Experiments mechanics of cell division and as many genes as we do—14,000 where we want to learn about cell polarity to genes compared to our 21,000 how embryos develop, or how developmental biology, behavior, genes. Even more impressive, over animals age, or how we learn, for and disease. 65% of human disease-associated example. Experiments where we The protagonist in this book is genes have a correlate (homolog) in want to test the limits of genetic Rosy (one of the Drosophila (Ugur et al., 2016). This engineering. first mutants discovered) and means that you can use Drosophila an homage to to learn about many of the genes the inspirational 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 that you have to take care of them. “overpopulation catastrophe”. If L1 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 Topics covered in this develop from fertilized eggs in Wa n d e r i n g L 3 9–12 days. This is great because it Chapter: means you can get lots of flies fast.