8.) MATERIALS & METHODS

8.1 Flystocks and Staging

Oregon-R was used as a wild-type strain. Embryos were raised at 25C and staged according to (CAMPOS-ORTEGA & HARTENSTEIN, 1997), if not stated otherwise. The age of specimens was referred to in hours after egg laying (AEL) at 25C. Precise staging was achieved by selecting embryos immediately after formation of the second midgut constriction (3-part-gut at 13 hr AEL +/- 10 min at 25C) and subsequent incubation for further time at 25C. Mutations were maintained over standard balancers with GFP markers.

To visualize muscles, I used w;G203zcl2144 that expresses GFP associated with the muscle protein titin (MORIN et al., 2001). Experiments involving motorneurons were done in animals of the HB9-Gal4 line (ventrally projecting neurons; BROIHER & SKEATH, 2002); OK371-Gal4 (full pattern of motorneurons; K.G. Moffat, J.B Connolly, J. Keane, S.T. Sweeney, and C.J. O'Kane, unpublished; see also MAHR & ABERLE, 2005); RN2-Gal4 (primarily aCC, pCC and RP2; LANDGRAF et al., 2003); and CQ-Gal4 (LANDGRAF et al, 2003). The line ST27-Gal4 (Stefan Thor, unpublished) was shown to be specific for U1 in all segments of the VNC using evenskipped as a marker. Experiments in the mesoderm were done in animals of the 24B-Gal4 (BRAND & PERRIMON, 1993). The effects of an apoptosis deficiency were investigated in H99 embryos (PETERSON et al., 2002), Abd-B null mutations in Abd-BM1 (SANCHEZ-HERRERO et al., 1985). Abd-B was ectopically driven by UAS-AbdBm (CASTELLI-GAIR et al., 1994). Single labelled motorneurons were generated by crossing OK371-Gal4 flies to flies of: heatshock flipase; UAS-Stop-CD8-GFP/Cyo; Tm6/MKRS (WONG et al., 2002). Single cells of the RN2 pattern were investigated in RN2-flp, tub-frt-stop-frt-Gal4, UAS-mCD8GFP (see PIGNONI & ZIPURSKI, 2001 for tubulin-CD2 stock; Annemarie North, unpublished).

Non-Drosophila Flies: Megaselia scalaris and Clogmia albipunctata were provided by Pat Simpson. Megaselia were kept in bottles. Larvae were obtained from laying pots that provided the flies with a yeast-glucose medium as in standard procedures for Drosophila. Clogmia was kept in a cage provided by Pat Simpson and fed with nettle powder as described elsewhere (ROHR et al., 1999). Larvae of both species and embryos of Megaselia were dissected and treated with standard procedures established for Drosophila.

8.2 Dechorionation and Wholemount Fixation

Embryos were harvested from agar plates and attached to laying pots. The eggs were exposed to bleach from a stock solution for about one to three minutes and washed several times in a cage with tap water in order to remove the chorion. Then the embryos were transferred into a glass vial with 500 mL heptane, 400 mL PBS (0.075 mL, pH 7.2) and 100 mL formaldehyde (stock solution at 40%), covered and kept under rotation for approximately 30 minutes. Then the embryos were transferred again into a 2 mL Eppendorf vial with 600 mL methanol and 600 mL heptane, using a pipette. They were vortexed for one minute in order to remove the vitelline membranes and washed in methanol for 3 times, approximately 15 minutes each. These washing steps were followed by four to five more with PBS-Tx (0.3%, 0.075 M, pH 7.2).

8.3 Immunohistochemistry

Fixed and washed specimens were blocked in PBS-T/BSA (0.075 M phosphate buffered saline at pH 7.2 with 0.3% triton-X and 5 % bovine calf serum) for 15 minutes. Then they were incubated with the primary antibody in phosphate buffered saline (0.075 M, pH 7.2) with 0.3% Triton-X overnight at 25C. After this, the antibody solution was removed and the specimens were rinsed in blocking solution (PBS-T/BSA) three to five times, 10 minutes each.

Primary Antibodies: rabbit a myosin (1:500; KIEHART & FEGHALI, 1986); Cy2-conjugated mouse a horseradish peroxidase (1:200; Jackson ImmunoResearch); mouse a fasciclinII (mAb 1D4; 1:200; VAN VACTOR et al., 1993); rabbit a evenskipped (1:500; FRASCH et al., 1987); mouse a engrailed (1:50; PATEL et al., 1989); mouse a GFP (1:500; Molecular Probes); mouse a connectin (1:50; Molecular Probes); rabbit a vestigial (1:200; WILLIAMS et al., 1993); rabbit a S59 and rat a S59 (1:200 each; CARMENA et al., 1995) guinea pig a Krppel (1:200; KOSMANN et al., 1998); rabbit a Lucifer Yellow (1:1000; Molecular Probes); rabbit a caspase-3 (1:50; Cell Signalling Technologies); mouse a Abdominal-B (1:200; CELNIKER et al., 1989); mouse a Antennapedia 4C3 (1:200; Developmental Studies, Hybridoma Bank); mouse a Ultrabithorax (1:200; WHITE & WILCOX, 1984); rabbit a teashirt (1:200; NG et al., 1996); mouse a abdominal-A (1:200; KELLERMANN et al., 1990); mouse a extradenticle (1:200; GONZALEZ-CRESPO & MORATA, 1995); rat a tiptop (1:200; LAUGIER et al., 2005); mouse a disco (1:100; ROBERTSON et al., 2004 ).

Secondary Antibodies: Specimens were incubated with the secondary antibody (either biotinylated for DAB: Vector Laboratories, 1:200; or fluorophor-conjugated for fluorescence microscopy: Alexa488, Alexa568, Alexa633; Molecular Probes, 1:200) for one hour at room temperature and then rinsed in saline three to five times. For fluorescence microscopy, specimens were mounted in glycerol-based VectaShield medium (Vector Laboratories). For DAB treatment, specimens were exposed to the ABC kit (Vector Laboratories; 1:100) for 20 to 40 minutes at room temperature. Specimens were finally rinsed three more times in PBS and transferred into a watchglass for DAB development.

8.4 DAB Development

15 mL DAB were solved in 300 mL PBS (0.075 M, pH 7.2). The specimens in a watchglass were exposed to about half this volume for 5 minutes to allow the DAB to penetrate the tissue. In the meantime, 5 mL of 30% H202 were diluted in 100 mL PBS and 10 mL of this solution added to the remaining DAB. For black precipitate with enhanced contrast, 10 mL of aqueous 8% NiCl solution were mixed with the DAB solution additionally. The final solution was added to the watchglass and the progress of the development was checked under a stereo microscope and stopped with PBS. The tissues were then rinsed three times in PBS. Specimens were finally mounted using DPX or more often Araldite.

8.5 Dehydration and Mounting

Specimens that were previously treated (wholemount fixation or flat preparation and immunohistochemistry) were dehydrated in ethanol (30%, 50%, 70%, 90%, 100%, 100%, 100%; approximately 10 min each) and either cleared with histoclear (15 min) for DPX, or directly mounted in Araldite. Slides with Araldite were polymerised overnight at approximately 60 C (on a hotplate).

8.6 Flat Preparations

Embryos were collected, dechorionated and staged as described in (LANDGRAF et al., 1997). Under phosphate buffered saline (0.075 M phosphate buffer, pH 7.2) the embryos were transferred to polylysine-coated coverslips, lifted out of the vitelline membrane with a glass needle, cut open along the body-axis (typically along the dorsal midline) and then attached to the coverslip. Using a glass needle that was attached to a mouth-piece (a plastic pipette tip) and a rubber tube, the gut and fat body were removed by gentle suction and the embryos were flattened by blowing a stream of saline over them. This way, the body wall was opened like a book and the CNS was exposed. The trachea were lifted up and either pinned away from the specimen or removed; the same treatment was applied to the hindgut. Flat preparations were fixed in 3.7% formaldehyde for 3 (dye-fills) to 30 (antibody labelling) minutes and rinsed in PBS (dye fills) or PBT (antibody labelling).

8.7 Microscopy and Imaging

Specimens were analysed on a Zeiss Axiophot Photomicroscope using transmitted light and incident-light fluorescence. Images and movies were collected using the Zeiss AxioCam Mrm CCD camera and Zeiss Axiovision 4.0 imaging software. Confocal Microscopy was performed on a Leica SP1 using 63x oil or water immersion objectives. In some cases, overlays or z-stack-projections were generated and processed with ImageJ (United States National Institute of Health software, Bethesda, Maryland, US) or Adobe Photoshop software (Adobe Systems Incorporated, San Jose, California US).

8.8 Dye Fills and Injections

DiI backfills of NMJs: Lipid-soluble carbocyanoine dye 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanide perchlorate (DiI) (Molecular Probes, Eugene, Oregon, US) was dissolved in vegetable oil as described by (BOSSING & TECHNAU, 1994) and backfilled into sharpened glass capillaries, which were then bevelled and mounted to a piece of rubber tubing and a plastic syringe. With use of a 63x water immersion lens on a Zeiss fixed-stage microscope or an Olympus BX-50-WI and a hydraulic micromanipulator (Narishige, Tokyo, Japan), a small droplet of DiI was manually deposited on a particular neuromuscular junction (NMJ) by applying pressure on the syringe. If necessary, motor neuron axons and NMJs were highlighted with a GFP conjugated anti-HRP antibody and the drop of dye was applied under UV light. Then the dye was left to diffuse overnight at 4C. To access NMJs of external muscles, internal muscle layers were removed surgically using a glass needle. Preparations were analysed using a Zeiss Axiophot microscope with UV (see 8.7 Microscopy and Imaging).

DiI Backfills of Nerves: To backfill the transverse nerve, Lipid-soluble carbocyanoine dye 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanide perchlorate (DiI) (Molecular Probes, Eugene, Oregon, US) was backfilled into microelectrodes of the same kind as the ones used for Lucifer Yellow injections (60-100 MW). The shaft was backfilled with 0.2 M LiCl. The transverse nerve was located using an Olympus BX50-WI fixed-stage microscope with a 60x water immersion lens with Nomarski optics and a hydraulic micromanipulator (Narishige, Tokyo, Japan). Upon application of a 0.8 to 1.5 nA DC current, a crystal formed on the tip of the electrode, which could be directly attached to the transverse nerve in the medial area of the VNC. Then the dye was left to diffuse overnight at 4C. Preparations were finally analysed using a Zeiss Axiophot microscope with UV (see 8.7 Microscopy and Imaging).

Lucifer Yellow Injections: For intracellular dye injection, microelectrodes (60-100 MW) were backfilled with 5% Lucifer Yellow solution (Molecular Probes). The shaft was backfilled with 0.2 M LiCl. Motorneuron cell bodies were penetrated using an Olympus BX50-WI fixed-stage microscope with a 60x water immersion lens with Nomarski optics and a hydraulic micromanipulator (Narishige, Tokyo, Japan). Lucifer Yellow was injected by the application of a 1.3 nA DC hyperpolarising current for two to five minutes using an Iontophoretic Dye Marker (Digitimer). Preparations were subsequently washed with saline (0.075 M phosphate buffered saline, pH 7.2), then fixed with 3.7% formaldehyde saline for 30 min at room temperature and washed once more. The specimens were then processed immunohistochemically to amplify the signal and mark reference cells.

8.9 Phalloidin Injections

1 mL of phalloidin (Alexa Fluor 568 phalloidin solution in 1.5 mL methanol) was mixed with 100 mL of a solution of 7.4% formaldehyde in phosphate buffered saline (0.15 M, pH 7.2) and 100 mL methanol. This resulted in a final solution of 1 mL phalloidin in 3.7% formaldehyde in PBS (0.075 M, pH 7.2) and 50% methanol (1:200). The methanol had proved to be crucial to permeabilise the muscle sheath. This solution was sucked into glass capillaries that were connected to a 10 mL syringe through a piece of rubber-tubing. The tip of this capillary was then pierced through the bodywall of third instar larvae on the dorsal midline approximately at segment A5 or A6 to get as close to the posterior end as possible without distorting the muscles in A8/9. A small amount of the phalloidin solution was injected, so that the larva expanded. The pressure was kept for 30 seconds, after which the capillary was retracted and most of the phalloidin ran out of the larva. This procedure was repeated two or three times and the larva was then examined under fluorescence (see Material and Methods 8.7).

8.10 Larval Preparations: Glue preparations and CNS Dissections

Larvae were anaesthetised by cooling them at -20C for about 5 minutes. Then they were transferred to a Sylgard (Dow Corning)-coated coverslip and fixed at both their anterior and posterior ends, using cyanoacrylate glue (Histoacryl; Braun, Melsungen, Germany) under phosphate buffered saline (0.075 M, pH 7.2) (BAINES & BATE, 1998). Larvae were then opened dorsally using sharp tungsten needles and glued flat to the sylgard coverslip. Gut and fat body were removed by sucking and blowing saline through a glass capillary attached to a short piece of rubber tubing in order to expose the ventral nerve cord and the muscle field. The tracheae were pulled off and glued to the sylgard. The specimen was then viewed using a 63x water immersion lens combined with Nomarski optics (see also 8.7 Microscopy and Imaging).

For simple CNS dissections of first to third instar larvae, specimens were transferred from apple juice agar plates into phosphate buffered saline (0.075 M, pH 7.2). The most anterior segments were removed and the CNS was dissected out with hypodermic needles. The CNS was then either transferred onto a poly-lysine coated coverslip or into a 500 mL Eppendorf vial and fixed in 3.7% formaldehyde dissolved in saline (0.075 M, pH 7.2) at room temperature for 15 min (first instar) to 30 min (third instar). Finally, the specimens were washed three times (10 min each) in saline and processed immunohistochemically or examined right away microscopically.

8.11 Heatshocks and Generation of MN-clones

OK371-Gal4 (K.G. Moffat, J.B Connolly, J. Keane, S.T. Sweeney, and C.J. O'Kane, unpublished; see also MAHR & ABERLE, 2005) flies were crossed with flies carrying the heatshock-Flipase and UAS-FRT-STOP-FRT-CD8-GFP constructs (heatshock flipase; UAS-Stop-CD8-GFP/Cyo; Tm6/MKRS) (WONG et al., 2002). Embryos were heatshocked on an apple juice agar plate at 37C for 30 min to generate a fractionated OK371 pattern with individual, identifiable motorneurons. The heatshocked embryos were raised on the apple juice agar plates with yeast at 25C and examined as third instar larvae by simply anaesthetising them (cooled at -20C for about 10 minutes) and transferring them into a drop of saline under a coverslip (see also 8.7 Microscopy and Imaging).

In order to visualise individual cells of the RN2 pattern, virgins of a RN2-flp, tub-frt-stop-frt-Gal4, UAS-mCD8GFP (see PIGNONI & ZIPURSKI, 2001 for tubulin-CD2 stock; Annemarie North, unpublished) were crossed to males of Oregon-R to improve their reproductive performance. In this line, the gene for the flipase is placed under the control of a weak evenskipped promoter. Flies were kept at 25C in a laying pot overnight. The apple juice agar plate was removed the next morning and embryos at a variety of stages incubated at 29C for 24 hours. The larvae were then raised at 25C and examined as third instars.

Flip-out events had occurred only in a subset of cells in the RN2 pattern, leading to Gal4 expression under the control of the Tub-promoter. Third instar larvae were anesthetised by cooling them at -20C for about 10 minutes. They were then transferred into a drop of saline and examined under a coverslip with UV. Larvae in which single cells were visualised were dissected and the CNS fixed and stained with antibodies against evenskipped and GFP (see 8.10 Larval Preparations; 8.3 Immunohistochemistry; and 8.7 Microscopy and Imaging).

 

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