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Arthropod/Fly Lab 2009 June 22-27 Arthropod/Fly Lab 2009 June 22-27 Lectures: Nipam H. Patel [email protected] (Mon & Fri) Steve Small [email protected] (Tue) Matt Ronshaugen [email protected] (Wed) Andrea Brand [email protected] (Thur) Labs: Andrea Brand [email protected] Cassandra Extavour [email protected] Pan-Pan Jiang [email protected] Henrique Marques-Souza [email protected] Nipam H. Patel [email protected] Matthew Ronshaugen [email protected] Page 1 of 80 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. Possible “Projects” 1) Segmentation Look at the mutant cuticle phenotypes of Drosophila segmentation mutants. Examine the expression patterns of segmentation genes in Drosophila (protein and mRNA), and compare them to the patterns of orthologous genes in orther arthropods (for example, examine engrailed and even- skipped expression in multiple species) Carry out live imaging of GFP lines for segmentation genes in Drosophila 2) Hox genes Look at the effect of Hox gene misexpression in Drosophila using the Gal4 UAS system Examine the expression patterns of Hox genes in Drosophila (protein and mRNA), and compare to Hox gene expression in other arthropods. Look at the expression of non-coding mRNAs from the Hox complex 3) Neurogenesis Look at the process of neurogenesis in Drosophila using various antibodies Look at mutants that effect fly neurogenesis Compare neurogenesis in multiple arthropod species. Examine neurogenesis in the fly eye disk 4) Axonogenesis Look at patterns of axonogenesis in Drosophila Examine mutations causing defects in axonogenesis Watch living neurons pathfind to their targets Examine the pattern of motoneuron connectivity with muscles in larvae, and axonogenesis in the eye disk. Compare axonogenesis patterns in multiple species 5) Dorso-ventral patterning Look at Drosophila mutations that effect D/V patterning Look at the expression of genes involved D/V patterning in Drosophila and compare to expression patterns in other arthropods. Look at the role of single-minded in Parhyale D/V patterning Page 2 of 80 6) Organogenesis Watch organogenesis in living Drosophila embryos using GFP expression Look at the expression pattern of genes involved in patterning organs such as somatic miscles and the heart and compare this to the patterns you see in other arthropods. 7) Appendage/Disk development Look at the development of Drosophila imaginal disks. Compare patterns of gene expression between Drosophila imaginal disks, the imaginal wing disks of butterflies, and the appendages of arthropods that develop directly without imaginal disks. Use Gal4 UAS system to lineage trace cells from the embryo into the disks 8) Germline/ovaries Examine the specifications, development, and migration of the germline in various arthropods. Examine ovary development in Drosophila and compare to ovary development in Triboloium, Oncopletus, and Schistocerca. Page 3 of 80 II. SCHEDULE Monday, June 22 Lecture: Nipam Patel: Intro to Arthropod Development Afternoon and Evening Labs: Arthropods/Flies 1 and 2 Goals: Start antibody stains on Drosophila embryos Be able to stage and navigate the Drosophila embryo Plan general experiments for the rest of the week Start fly crosses and in situs Tuesday, June 23 Lecture: Steve Small Afternoon and Evening Labs: Arthropods/Flies 3 and 4 Goals: Continue antibody and in situ stains on Drosophila embryos Learn to dissect many other arthropods (and start staining!) Wednesday, June 24 Lecture: Matt Ronsgaugen Afternoon Lab: Arthropod/Flies 5 plus Plankton Tow/Outdoor Collecting (snorkeling!) Evening Lab: Arthropod/Flies 6 Goals: Find embryos to examine in Plankton Tow or from your own collecting Continue other experiments Thursday, June 25 Lecture: Andrea Brand Afternoon and Evening Labs: Arthropods/Flies 7 and 8 Goals: Continue working on dissections, injections, stainings Friday, June 26 Lecture: Nipam Pateln Afternoon and Evening Labs: Arthropods/Flies 9 and 10 Goals: Continue working on dissections, injections, stainings Saturday, June 27 Lecture: Richard Harland Afternoon Lab: Arthropods/Flies 11 Goals: Finish Arthropod/Fly projects and get ready for Show and Tell Evening: Show and Tell!!! Page 4 of 80 III. Experimental Details Section 1. Antibody Staining a. Drosophila 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. Each group should complete all seven of the following antibody stains on Drosophila. You will be using a “Rapid” version of the standard antibody. Your fly embryos are a mix of embryos ranging from 0-17 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. Your aliquot should last you throughout this and future experiments during the Arthropod module. Additionally, too much tissue will give a decrease of signal! React Antibody Staining Pattern µl:300 1:3000 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 Goat anti-mouse regional expression pattern Ni HRP (115-035-003) DAB+ 3) BP102 Stains all CNS axons (unknown antigen) 1.5 Ni 4) BP102 See above 1.5 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 7) Goat anti-HRP-FITC 2.0 NO SECONDARY flour Page 5 of 80 While this protocol produces antibody stains in one day, it only works well on very robust antibodies. The “usual” protocol can be found further down in this manual. 1. Rehydrate with 2X 1 min followed by 1X 10 min with PT. 2. Incubate 10 min in 100 µl PT+NGS. 3. Add primary antibody to the appropriate final concentration. Primary antibodies may be used at about 1.5 to 2 times the “normal” concentration (i.e., that used for the regular staining procedure). 4. Mix and incubate in the primary antibody at room temperature for 30 min. 5. Wash 3X 1 min with PT. 6. Wash 3X 10 min with PT. 7. Add secondary antibody. It is not necessary to pre-block with PT+NGS. For HRP immunohistochemistry, use the goat anti-mouse IgG at a dilution of 1:250. Mix and incubate in the secondary antibody for 30 min at room temperature. 8. Wash 3X 1 min with PT. 9. Wash 3X 10 min with PT. 10. React with the appropriate reaction protocol. (See general antibody prootocol, IV.1) 11. Wash with PT. 12. Start your next staining (if doing multiple labels). If you are done, put the embryos into 200µl 50% glycerol with DAPI for 10 – 30 min and then into 300 µl of 70% glycerol. The embryos will be fine in glycerol for several weeks at room temperature, several years at 4°C, and several decades at –20°C. Staining may fade (even over just a few hours) if your glycerol solution is acidic. Page 6 of 80 Into the wild Use the protocol and expertise 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 mellifers (honeybee) Oncopeltus fasciatus (milkweed bug) Crustaceans: Parhyale hawaiensis (beach hoppers—amphipod crustaceans) Gammarus sp. (beach hoppers—amphipod crustaceans) Orchestia species Mysidium columbiae (opossum shrimp) Triops longicaudatus (tadpole shrimp) Artemia salina (brine shrimp or “sea monkeys”) Marmokrebs. (marble crayfish) Chelicerata: Parasteatoda tepidariorum (common house spider) Page 7 of 80 Molecular markers of embryonic development c.1. 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 (and 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) on Parhayle 7) Double label 2B8 (Black, the rapid protocol in the protocol section will work on this) + 1G10 (Brown) on Drosophila 8) Double label
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