Arthropod Lab Part1: Flies Nipam Patel [email protected] Matthew Ronshaugen [email protected] Justin Bosch [email protected] Katherine Brown [email protected] Heather Bruce [email protected] Erin Jarvis [email protected] Ryan Null [email protected] TABLE OF CONTENTS I. INTRODUCTION 2 II. SCHEDULE 3 III. EXPERIMENTAL OVERVIEW 4 IV. PROTOCOLS 9 IV.1 General Fixation and Antibody Staining 9 IV.2 Rapid Antibody Staining Protocol 11 IV.3 Fixing and Staining Post-embryonic Tissues (Imaginal Discs) 12 IV.4 In situ Hybridization 14 IV.5 EdU labeling of cell proliferation in tissues 16 V. GENERAL SOLUTIONS 17 V.1 Fixatives 17 V.2 Solutions for antibody protocols 17 V.3 Solutions for in situ hybridization 19 VI. MAKING DISSECTIONS TOOLS 20 VI.1 Making blunt probes 20 VI.2 Making Tungsten needles for dissecting 20 VII. AVAILABLE STOCKS & REAGENTS 21 VII.1 Antibodies Separate Handout VII.2 In situ Probes 21 VII.3 Fixed Embryos 26 VII.4 Drosophila Stocks 27 VIII. DROSOPHILA DEVELOPMENT & STAGING 28 VIII.1 Embryogenesis 28 VIII.2 Larval Development and Morphogenesis 32 VIII.3 Adult Morphology 34 VIII.4 Female Reproductive System 35 VIII.5 Male Reproductive System 37 VIII.6 Imaginal Disks and Generation of Mosaic Tissues 38 1 I. Introduction In this module, you will learn about a variety of arthropod systems, including the model genetic system, Drosophila melanogaster. Most importantly, we would like you to leave with the ability to analyze and compare the development of different arthropod embryos. In order to do that, you will be performing various molecular and embryological techniques, such as antibody staining, in situ hybridization, live imaging, and lineage tracing using both classical injection and modern genetic methods. At first we would like you to use a set of antibodies to detect the expression of important developmental proteins and RNAs in the fruit fly Drosophila melanogaster. This will allow you to master the procedure of antibody staining, examine the spatiotemporal expression of these proteins, and study the development of various tissues. Because many of these proteins are strongly conserved in all arthropods studied to date, a subset of these antibodies allow you to go crazy and stain all sorts of arthropods that cross your way!! We will have many critters from which you can collect embryos and make them shine! In this module you will also have the opportunity to look at aspects of post-embryonic development. In particular, we will look at wing imaginal disc development in Drosophila and scale patterning in butterflies. You can stain imaginal disks with available antibodies and compare the expression pattern of various proteins between flies and butterflies, and look at butterflies scales by scanning electron microscopy. We will also give you several ideas for “projects” that we can discuss on the evening of the first day of the lab, but we also encourage you to try out as many techniques as possible and to look at as many critters as you can. We have provided an extensive list of possible experiments in section III. We suggest you read through them all and see what captures your interest. Do not try to do too much; by no means do we think you can or should do all of them. So be selective to optimize your time here. Talk to use about your goals, and we will help you plan and execute your experiments. 2 II. Schedule Monday (lecture: Chris Rushlow) Begin rapid staining Look at pre-stained Drosophila embryos (demo of mounting) Review of available antibodies Complete and begin imaging rapid stains Dissection of Drosophila imaginal disks (demo at 8 PM) Set up overnight staining, live imaging, and embryo collections Tuesday (lecture: Alan Spradling) Continue antibody staining Introduction to in situs Volocity training (2:30 – 4:00) Introduction to fly genetics and crosses Parhyale and Mysid dissection (demo at 5 PM) Ovary dissection (demo at 8 PM) Wednesday (lecture: Iswar Hariharan) Arthropod diversity (inc. plankton tow; demos at 8 PM) Parhyale injections (demos at 2PM) Dissection of butterfly imaginal disks (demo at 4 PM) Thursday (lecture: Nipam Patel) Continue experiments Softball practice or snorkeling (weather permitting) Friday (lecture: Matt Ronshaugen) Continue experiments Saturday (lecture: Nipam Patel) Continue experiments Show N’ Tell 3 III. Experimental Overview III.1 Observations of Embryogenesis A. RAPID Antibody Staining of Drosophila Embryos. In this experiment, you will investigate protein expression patterns throughout Drosophila development that illustrate the following gene classes/organs (appropriate antibody in parentheses): maternal gradient (Rb anti-Bcd), pair-rule (Rb anti- Ftz, Rb anti-Eve), segment polarity (M anti-wingless [4D4]), Hox (M anti-Antp [8C11], Rb anti-teashirt), CNS (anti-HRP), and muscle (Ratb anti-tropomyosin). During the Arthropod Module, you will see more examples of these patterns in Drosophila and examine whether other arthropods share them as well. To do the experiment, split into six groups of four. Each group should complete all four of the assigned antibody stains on Drosophila, plus one control tube (no primary antibody). The rapid antibody protocol is in section IV of this manual, but is repeated below. Use the primary antibodies at the "60 min" dilutions listed on the antibody table. Use secondaries at 1:500 (as opposed to the standard 1:1000). NOTE: This rapid protocol only works for a few antibodies. Most every antibody will work better using the standard protocol (overnight in primary, 2 hrs in secondary). The fly embryos you will be given are a mix of embryos ranging from 0-18 hours and are in methanol. The tube contains enough embryos for the five stains you will do here. When carrying out fly embryo staining in eppendorf tubes, plan on having 15µl of settled embryos in MeOH per 1.5ml eppendorf tube. This will be about 20µl when rehydrated. Using too many or too few embryos can affect the signal-to- noise ratio of your staining. 1. Rehydrate 3X 1 min followed by 1X 10 min with PT. 2. Incubate 10 min in 200 µl PT+5% NGS. 3. Add primary antibody to the appropriate final concentration. ("60 min" concentration) 4. Mix and incubate in the primary antibody at room temperature for 60 min. 5. Wash 3X 1 min with PT. 6. Wash 3X 10 min with PT. 7. Add secondary antibody (in 200 µl of PT+ 5% NGS). It is not necessary to pre-block with PT+NGS. Use fluorescently labeled secondaries at a dilution of 1:500 when using this rapid protocol (as opposed to 1:1000 with the "normal" protocol). 8. Wash 3X 1 min with PT. 9. Wash 3X 10 min with PT. 10. Add 200 µl 50% glycerol with 1.0 µg/ml DAPI and incubate 15 min. 11. Remove 50% glycerol and add 70% glycerol (no DAPI). Embryo will be ready to view in about 15 minutes. 4 B. Molecular Markers of Embryonic Development Using a specific set of antibodies you will be able to see the expression of proteins involved in different steps of early development, such as the formation of the body segments, the specification of the different body regions (read, thorax and abdomen) as well as the formation of neurons and axons. We provide you with a list of additional antibodies for you to expand your expression analysis. You can either select a few genes and compare them across many different species or, pick a few arthropod species and study the expression of many genes. Gap and Pair Rule Look at the expression patterns of gap and pair-rule genes during early Drosophila development. Look at the expression of gap and pair-rule orthologs in other arthropods (you are encouraged to look in other phyla as well). Pair Rule and Segment Polarity Examine the expression patterns of Pax 3/7 and Even-skipped orthologs in a variety of organisms. How does this compare with the expression of Engrailed orthologs across species? What does this say about the evolution of segmentation in arthropods? Hox and Appendage Formation Examine the expression of Hox genes in Drosophila and other arthropods. Examine how Hox gene patterns have changed during evolution and the possible morphological consequences of these changes. Neurogenesis and Axonogensis Examine the process of Drosophila neurogenesis and axonogenesis. Compare neurogenesis and axonogenesis in Drosophila to neurogenesis and axonogenesis in other arthropods C. Live Imaging of Drosophila Embryos Collect Drosophila embryos at the appropriate stage (as close to fertilization as possible if you want to look at early nuclear divisions; late blastoderm if you want to watch gastrulation, mid germband extension for tracheal development, early germband shortened to view dorsal closure), mount in petri dishes with glass bottom coverslips coated with heptane glue, fill with Schneider's insect media (room temp) and image away on confocal or inverted widefield microscope. Try live imaging the following: 1) Wild type 2) GFP and RFP lines (e.g. moesin:GFP, histone:RFP, hh-GFP, Btl-GFP is particularly excellent) 3) GAL4 lines (e.g. Dpp-Gal4, hh-Gal4) crossed with UAS-RFP or G-TRACE. D. GAL4 drivers in development, in live or fixed animals The GAL4/UAS binary system has been adapted from yeast for use in flies. Expression of the GAL4 gene is driven by a specific promoter (e.g. hsp70, tubulin, actin, nubbin, etc.). GAL4 binds to Upstream Activating Sequences (UAS) and activates transcription of genes adjacent to theses sites. This allows for any gene placed downstream of UAS to be expressed in a specific tissue or at a specific time point in development. In Drosophila a wide range of different GAL4 drivers and UAS transgenes are available.