Arthropod/Fly Lab 2010 June 21-26 Lectures: Celeste Berg Iswar Hariharan Nipam H. Patel Matthew Ronshaugen Labs: Bill Browne Elise Delagnes April Dinwiddie Abby Gerhold Yassi Hafezi Hannah Rollins TABLE OF CONTENTS I. INTRODUCTION 2 II. SCHEDULE 3 III. EXPERIMENTAL OVERVIEW 4 IV. PROTOCOLS 13 IV.1 General Fixation and Antibody Staining 13 IV.2 Rapid Antibody Staining Protocol 15 IV.3 Histochemical Development Reactions 16 IV.4 Labeling with Multiple Primary Antibodies 18 IV.5 Fixing and Staining Other Arthropods 19 A. Spider 19 B. Artemia 20 C. Mysids 21 D. Grasshoppers 22 E. Parhyale 23 IV.6 Fixing and Staining Post-embryonic Tissues (Imaginal Discs) 27 A. Drosophila Wing Imaginal Discs 27 B. Butterfly Wing Imaginal Discs 29 IV.8 In situ Hybridization 30 IV.9 Injection in Parhyale 33 V. GENERAL SOLUTIONS 36 V.1 Fixatives 36 V.2 Solutions for antibody protocols 36 V.3 Solutions for in situ hybridization 38 VI. MAKING DISSECTIONS TOOLS 40 VI.1 Making blunt probes 40 VI.2 Making Tungsten needles for dissecting 40 VII. AVAILABLE STOCKS & REAGENTS 41 VII.1 Antibodies Separate Handout VII.2 In situ Probes 42 VII.3 Fixed Embryos 43 VII.4 Drosophila Stocks 44 VIII. DEVELOPMENTAL BIOLOGY & ANIMAL STAGING 46 VIII.1 Parhyale 46 VIII.2 Drosophila 51 VIII.3 Spiders 65 VIII.4 Grasshoppers 67 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 and analyze mutant phenotypes. In order to do that, you will be performing different molecular and embryological techniques, such as antibody staining, in situ hybridization, live imaging, and lineage tracing. At first and most importantly, we would like you to use a set of antibodies to detect the expression of important developmental proteins in the fruit fly Drosophila melanogaster. This step will allow you to master the procedure of antibody staining while studying the spatiotemporal expression of these proteins. Because these proteins are strongly conserved in all arthropods studied to date, this set of 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. You can stain these tissues with available antibodies and compare the expression pattern of various proteins between flies and butterflies. We will also conduct a clonal analysis experiment in which we will ask whether certain mutations affect the sorting, positional or adhesive properties of cells with the developing wing disc. We 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. 2 II. Schedule Monday (lecture: Intro to Embryogenesis and Post-embryonic Development of Drosophila) Look at pre-stained embryos Rapid staining Review of available antibodies, in situ probes, and fly stocks Dissection of imaginal disks Imaging 101A Tuesday (lecture: Growth Control) Parhyale injections Imaginal disk cell sorting experiment Other arthropods Chalk Talk Wednesday (lecture: Arthropod Evo-Devo) More arthropods (plankton tow) Ovary dissection Imaging 101B Thursday (lecture: Non-coding RNAs) Continue experiments Snorkeling (weather permitting) Friday (lecture: Oogenesis) Continue experiments Saturday (lecture: Butterfly wing patterning) Prepare for Show N’ Tell Show N’ Tell Sunday Whaling 3 III. Experimental Overview III.1 Observations of Embryogenesis A. Antibody Staining of Drosophila Embryos Antibody staining Drosophila will prepare you for staining other arthropods. In this experiment, you will investigate protein expression patterns throughout Drosophila development in the following gene classes (appropriate antibody in parentheses): pair-rule (DP312), segment polarity (DP312), Hox (FP3.3), and axons/neurons (BP102, 9F8, DP312). 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 along the length of each bench. Each group should complete all six of the following antibody stains on Drosophila. The rapid antibody protocol is in section IV (on page 15) of this manual. ** DO NOT USE ALL OF YOUR FLIES. These flies are a mix of embryos ranging from 0-16 hours. Only use 15µl of settled fly embryos in MeOH per 1.5ml eppendorf tube. This will be about 20µl when rehydrated. If you are unsure how many flies to use, ask for help. We will have examples showing a good amount of embryos to use. Using to many or too few embryos can effect the signal to noise ratio of your staining. React Antibody Staining Pattern µl:300 1:300 with Perform the following stains as rapid antibody stains (1-day) Anti-Pax3/7 (pair-rule, segment polarity, neural DAB+ 1) DP312 gene family) In Drosophila, pair-rule pattern 10.0 Ni early, then segmental pattern, then CNS pattern Anti-Ubx (homeotic gene). In Drosophila, DAB+ 2) FP3.3 15.0 regional expression pattern Goat anti-mouse Ni HRP conjugated DAB+ 3) BP102 Stains all CNS axons (unknown antigen) 5 Ni 4) BP102 See above 5 AEC Stains nuclei of all neurons (elav gene product, DAB+ 5) 9F8 15.0 encodes neural-specific splicing factor) Ni Goat anti-mouse AP BCIP/ 6) 9F8 See above 15.0 (115-055-166) NBT 4 B. Into the Wild – Comparative Antibody Staining in Other Arthropods Use the protocol and expertise you have acquired from your Drosophila antibody stains to detect the expression of these developmental proteins in many different arthropod species. Besides the additional arthropods listed below, you can collect specimens from the area around Woods Hole. Enjoy! Insects: Junonia (Precis) coenia (buckeye butterfly) Tribolium castaneum (flour beetle) Schistocerca americana (grasshopper) Apis millifera (European honeybee) Crustaceans: Parhyale hawaiensis (amphipod - beach hopper) Chelura sp. (amphipod - beach hopper) Caprella sp. (amphipod – skeleton shrimp) Limnoria sp. (isopod) Cyathura sp. (isopod) Libinia sp. (spider crab) Pagurus longicarpus (long-claw hermit crab) Emerita talpoida (mole crabs) Crangon septemspinosa (sand shrimp) Palaemonetes sp. (grass shrimp) Mysidium columbiae (opossum shrimp) Balanus sp. (barnacle) Artemia salina (brine shrimp - “sea monkeys”) Chelicerata: Parasteatoda tepidariorum (common house spider) Limulus polyphemus (horseshoe crab) Tanystylum sp. (pycnogonid - sea spider) Callipallene sp. (pycnogonid - sea spider) Some suggestions: 1) Examine the expression of Engrailed, Pax3/7, FP6.87, and the pattern of axonogenesis with αHRP at various stages in an arthropod of your choice. 2) Do a double antibody stain with Pax3/7 plus Engrailed and/or Ubx/abdA plus Pax3/7 in Parhyale or any other arthropod of your choice. What is the pattern and how does it compare to Drosophila? In Parhyale, can you identify the different rows in which each gene is expressed? 5 C. 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). Stains to do and compare: 1) 1G10 (anti-Hunchback) on Drosophila 2) 7C11 (anti-Hunchback) on Grasshoppers (15 - 25%) 3) 2B8 (anti-Eve) on Drosophila, Tribolium and Mysids stage 1 and 2 (sonicate stage 2) 4) 3B9 (anti-Eve) on Grasshopper (15-25%) 5) 7H5 (anti-Eve) on Artemia (sonicated) 6) RαPhEve (anti-Eve, ask Nipam for antibody) on Parhyale 7) Double label 2B8 (Black, the rapid protocol in the protocol section will work on this) + 1G10 (Brown) on Drosophila 8) Double label 7C11 (Black) + DP312 (Brown) on Grasshoppers (15-25%) 9) Double label 2B8 (Black) + 4D9 (Brown) on Tribolium 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? Stains to do and compare: 1) DP312 (anti-Pax 3/7) on Drosophila, Tribolium, Oncopeltus, Grasshoppers (15 – 30%), Parhyale, Mysids, stage 1, 2, and 3 (sonicate stage 2 and 3), Artemia (sonicated), Spiders, and Limulus 2) 2B8 (anti-Eve) on Drosophila, Tribolium, Mysids stage 1 and 2 (sonicate 2) 3) 3B9 (anti-Eve) on Grasshopper (15-25%) 4) 7H5 (anti-Eve) on Artemia (sonicated) 5) RαPhEve (anti-Eve) on Parhyale 6) 4D9 (anti-engrailed) on Drosophila, Grasshoppers (15 – 30%), Tribolium, Parhyale, Mysids, stage 1,2, and 3 (sonicate stage 2 and 3) 7) 4F11 (anti-engrailed) on Artemia (sonicated) 8) Double label DP312 (Black) + 4D9 (Brown) on Drosophila, Tribolium, Grasshoppers, and Parhyale 9) Double label DP312 (Black) + 4D9 (Brown) on Mysids stage 1 and 2 (sonicate stage 2) 10) Double label DP312 (Black) + 4F11 (Brown) on Artemia (sonicated) 6 Hox and Appendage Formation Examine the expression of Hox genes in Drosophila and other arthropods.