About the Dataset

• The Credits
• About the Datasets
• Contact
• Strain Full Name
• Publications
• Materials
• Method
• Notes
• RNA in-situ hybridisation using a robot
• Slide Analysis

The Credits

This Drosophila testis gene expression dataset was generated between 2003 and 2008 by Elizabeth Benson, Elin Gudmannsdottir and Helen White-Cooper in the Department of Zoology at the University of Oxford, with funding from the UK's BBSRC.

About the datasets

This dataset currently contains 1,947 images and metadata relating to 623 genes relevant to Drosophila spermatogenesis. Further images of additional genes will be added as they become available. For many genes, the PCR primer sequences used (derived from genomic sequences) and the predicted sequence of the PCR reaction are given. Sequences for other genes will be added at a later date. The PCR products were used to make the antisense RNA probes used for in situ hybridization.

Contact Information

Full Names of the Mutant Strains

Note: for wild type (wt) we also use the abbrevations red e or w[1118].

Publications

Carine Barreau, Elizabeth Benson and Helen White-Cooper, Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription, Biochemical Society Transactions. (2008) 36:540–542.

Carine Barreau, Elizabeth Benson, Elin Gudmannsdottir and Helen White-Cooper, Post-meiotic transcription in Drosophila testes, Development. 2008 Jun;135(11):1897-902.

Benson,E, Gudmannsdottir,E, Klyne,G, Shotton,D, White-Cooper, H., FLYTED - THE DROSOPHILA TESTIS GENE EXPRESSION DATABASE, 20th European Drosophila Research Conference, Vienna, 2007.

[Abstract] The making of mature sperm involves many processes including mitosis, meiosis and morphogenesis; unsurprisingly these involve a huge number of genes. The meiotic arrest genes activate expression of many target genes in the testes. Our Affymetrix array, comparing aly, comr, achi/vis, topi and can mutant testes to wild type testes, indicates that around 8500 genes are expressed in wild type testes, the majority of these genes are uncharacterised (CG number genes). The array also identified around 1000 genes that had significantly lower expression in all the meiotic arrest mutant testes compared to wild type testes, and around 450 genes that showed significantly higher expression in these mutant testes. From this array over 1000 genes have been semi-randomly selected, sampling genes with high/ medium/ low expression in wild type testes and up/ down/ unchanged expression in mutant testes, with a biased towards conserved genes. Using Q-RT-PCR and RNA in-situ hybridisation, the spatial and temporal distribution of these transcripts has been described in wild type testes and meiotic arrest mutant testes. The majority of genes that showed increased transcript levels in the meiotic arrest mutant testes were expressed early in spermatogenesis; the meiotic arrest genes may function to repress their continued expression in mature primary spermatocytes. Conversely, the majority of genes with reduced transcript levels in the mutant testes persisted into spermiogenesis and their expression in the testes was dependent on the function of the meiotic arrest genes. In addition, the meiotic arrest genes showed differential gene regulation; a subset of meiotic arrest dependent genes were significantly more dependent on achi/vis and/or topi then on the other meiotic arrest genes for their expression. All this data will be curated into a custom built fully-searchable database available on the web (www.fly-ted.org) for the whole research community.

Materials

1. Alkaline phosphatase - conjugated anti-digoxygenin antibody (Roche), pre-adsorbed against embryos, see note 5.
2. Ethanol series: 100%, 90%, 70%, 50%, 30%, in distilled water.
3. Fixative: 4% paraformaldehyde in 100 mM HEPES pH 6.9, 2 mM MgSO4, 1 mM EGTA. May be stored at -20℃, check pH before (re)use. (GLOVES!)
4. Glycine (Stock is 200 mg/ml, store at room temp)
5. GMM (Gary's magic mountant): 1.6 g/ml Canada balsam (powder) in methyl salicylate. If only liquid Canada balsam is available, mix 4:1 with methylsalicylate.
6. High pH Buffer (HP): 100 mM NaCl, 100 mM Tris pH 9.5, 50 mM MgCl2, 0.1% Tween 20: make up fresh, add MgCl2 last to prevent precipitation.
7. Hybridisation Buffer (HB): 50 ml formamide, 25 ml 20xSSC (salt + sodium citrate stock solution, 3 M NaCl, 0.3 M sodium citrate, pH 7.0), 1 ml of 10 mg/ml denatured sonicated salmon sperm DNA, 50 μl of 100 mg/ml heparin, 100 μl of Tween 20, 5 ml of 2 M citric acid, and 18.85 ml of water, final pH 4.5. Store at -20℃. Preheat for 65℃ washes. (GLOVES!)
8. Methyl salicylate (VENTILATION).
9. NBT (Nitroblue tetrazolium): 75 mg/ml in 70% dimethyl formamicle (DMF) stock (Roche).
10. PBST: PBS +0.1% Tween 20
11. Proteinase K (Roche, stock is 19 mg/ml, stored at -20℃)
12. Tissue culture plates (24-well) and tissue culture inserts with 8 μm mesh (Falcon #3097).
13. X-Phosphate substrate (BCIP; 5-bromo-4-chloro-3-indolylphosphate): 50 mg/ml in DMF stock (Roche).

Methodology

1. Dissect testes from young male Drosophila (0-1 day old) in testis buffer and transfer to 1.5 ml tube (note 1).
2. Fix 20-60 min. Agitation is not required, simply leave the tube on its side.
3. Wash 3x5 min in PBST.
4. Incubate in 50 μg/ml proteinase K in PBST for 5-7 min (note 2).
5. Stop the digestion with 2 mg/ml glycine in PBST for 2 min.
6. Wash 2x5 min in PBST.
7. Refix for 20 min as above (step 2).
8. Wash 3x10 min in PBST.
9. Wash in 1:1 PBST:HB for 10 min.
10. Wash in HB for 10 min.
11. Transfer into tissue culture inserts in a 24-well tissue culture plate (note3).
12. Prehybridise in HB at 65℃ for at least 1 hr (note 4).
13. Dilute the probe in 400 μl HB (I usually use 1-2% of a full scale Roche digoxygenin RNA reaction, i.e. I do a 20 μl reaction, hydrolyse if necessary, precipitate, resuspend in 200 μl water and use 2-4 μl of this), heat denature at 80℃ for 10 min, then briefly chill on ice.
14. Hybridise at 65℃ overnight.
15. Wash at least 6x30 min in HB at 65℃.
16. Wash 1x15 min in 4:1 HB:PBST at room temperature.
17. Wash 1x15 min in 3:2 HB:PBST at room temperature.
18. Wash 1x15 min in 2:3 HB:PBST at room temperature.
19. Wash 1x15 min in 1:4 HB:PBST at room temperature.
20. Wash 2x15 min in PBST.
21. Incubate overnight at 4℃ in preadsorbed (note 5) alkaline phosphatase - conjugated anti-digoxygenin antibody diluted 1:2000 in PBST.
22. Wash 4x20 min in PBST.
23. Wash 3x5 min in freshly made buffer HP.
24. Make staining solution in HP buffer by adding 4.5 μl NBT and 3.5 μl X-Phosphate per ml.
25. Add this staining solution to the testes and leave to develop in the dark. The signal typically takes 10 min-1 hour to develop, although for some transcripts incubation for several hours may be needed.
26. Stop the reaction by washing 3x5 min in PBST.
27. Dehydrate through an ethanol series. 10 min in each of 30, 50, 70, 90 and 100% (twice) ethanol. Transfer testes into a glass staining block (note 6)
28. Incubate 15 min in 1:1 ethanol: methyl salicylate, then in 100% methyl salicylate (note 7).
29. Mount in GMM and observe with Nomarski optics.

Notes

1. Prepare about 10 males per probe. If many probes are to be used, process testes together until the transfer into tissue culture inserts step. Removal of the accessory glands and other bits of genital tract is not essential: they can provide a nice in-sample negative control. To transfer testes, place a small drop of testis buffer in the lid of a 1.5 ml tube, put testes into this, add 600 μl of fix to the tube, close lid and mix. Testes stick to tweezers if you try to put them directly into fix.
2. New batches of proteinase K should be checked. Over-digestion results in testes which are very fragile, and which are sticky and tend to clump together.
3. To incubate at 65℃, the tissue culture dish can be left floating in a water bath. Add washes into inserts, remove by lifting up insert and aspirating solution from the well. Careful, sometimes the mesh at the bottom becomes detached: check for loose testes before aspirating!
4. Testes may be stored in HB at -20℃ for up to a week without loss of quality.
5. To preadsorb the antibody, fix embryos using a protocol suitable for immunostaining. Dilute the antibody 1:20 in PBST, and incubate with the rehydrated fixed embryos for 2 hr. Remove from embryos and store at 4℃.
6. To transfer stained testes, use a 200 μl pipette tip with the end cut off. Watch under the dissecting microscope to ensure complete transfer, especially of lightly stained tissue.
7. Methyl salicylate clears the testes, but also dissolves some of the colour product. Therefore this incubation will make background staining go away, but care must be taken to prevent the real staining from going away too. Canada balsam stabilises the colour, so remove the methyl salicylate and add GMM to the staining block when ready. The colour should look quite intense under the dissecting microscope, in order to get good higher magnification pictures. To mount, transfer in GMM onto a 22x22 mm2 coverslip. Methyl salicylate also makes the testes very brittle, so any dissection (e.g. separation of pairs of testes) can be done by prodding or cutting with a tungsten needle.

RNA in situ hybridisation using a robot

We use a BioLane HTI robot. The robot is pre-programmed with all the suitable steps from the manual protocol. However, different inserts and plates are used which are compatible with the robot. Steps 1-14 are done manually. The washing steps 15-23, 25 and 26, and the length of the antibody incubation, are controlled by the robot (we usually do a 12 hour incubation). The colour reaction is terminated manually. When the colour reaction in all the samples has been stopped, the PBST and ethanol washes (steps 26 and 27) are done by the robot. The slide mounting steps (steps 28 and 29) are done manually.

Slide Analysis

Slides are examined on a Olympus Bx50 upright microscope using DIC microscopy; typically at 10x magnification. Every testes on each slide is examined. Images are captured with a JVC KY-F75U three-colour CCD camera with KY-Link software, with no further manipulations. Pictures are taken of at least one wild type and one mutant testis that give the best representation of the staining pattern. Additional pictures are taken if the staining is "exciting", and higher magnification pictures are taken of interesting features. If staining occurs in somatic tissues, such as accessory glands, pictures of these are also taken. For all pictures, the following is noted: slide name, date taken, probe dilution in 500 μl of HB, genotype, position on slide, magnification, and the description of staining pattern.