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Using RNA interference mosaics to map retained mutant phenotypes

Using RNA interference mosaics to map retained mutant phenotypes HHMI Internship Megan Kelly Mentor: Dr. Barbara Taylor Dept. of Zoology www.berkeley.edu/.../07/images/fruitflies.jpg Background How do genes work in creating body parts and controlling function? I focused on

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Using RNA interference mosaics to map retained mutant phenotypes

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  1. Using RNA interference mosaics to map retained mutant phenotypes HHMI Internship Megan Kelly Mentor: Dr. Barbara Taylor Dept. of Zoology www.berkeley.edu/.../07/images/fruitflies.jpg

  2. Background How do genes work in creating body parts and controlling function? I focused on answering this question for the female fruitfly reproductive tract Ovaries Oviducts Uterus http://flybase.net/static_pages/imagebrowser/imagebrowser10.html

  3. One method for analysis Using mutation in general to disrupt gene expression causing mutant phenotype Changing gene expression in only specific cells to locate mutant phenotypes I altered (retained) gene expression in cells of the female and found that in resulting phenotypes no eggs were laid - a result of retention.

  4. Background retained(retn) gene in fruit flies plays role in female reproductive tract development Strong mutation, prevents the gene from transcription, therefore no expression. Results in embryonic lethal Weak mutation, (missense). Adults viable, results in female sterility, and abnormalities in courtship behavior.

  5. Hypothesis Female sterility is due to a retained mutant phenotype in the reproductive tract If true, females should be able to mate but not release an egg from the ovaries Wildtype reproductive tract http://flybase.net/reports/FBim0000078.html

  6. Goal Map the location of cells responsible for female sterility by comparing retn flies to retnRNA-interference mosaics Mosaics: Groups of cells in the body vary in gene expression Ex: Calico cat has variation in pigment due to genotypic variation in skin cells http://tigerpixie.com/tigerpixieart/HalloweenaSM.jpg

  7. Wildtype(CSA) retained(retn) Mosaic = retn expression location X

  8. UAS retn-RNAi hthgal4 UAS X gal4 gene homothorax gene retn-RNAi gene Gal4 protein gal4 gene homothorax gene retn-RNAi gene Creating mosaics retn-RNAi I retn-RNAi III retn-RNAi ARID

  9. Normal homothorax expression • homothorax • hthgal4 • UASretn-RNAi Normal retn expression • retn expressing • retn expressing • homothorax Loss of retn function • UASretn-RNAi • hthgal4

  10. Comparing retn mutants to our RNAi mosaics in three ways: • Behavior before and during copulation • Fertility http://www.pnas.org/misc/archive072803.shtml http://www.csus.edu/indiv/h/hollandb/Pictures/fruit%20flies/fly%20laying%20egg.jpg • Physical makeup of the reproductive tract through dissections http://flybase.net/reports/FBim0000078.html

  11. Latency to courtship Latency to copulation Copulation duration Behavioral test Wildtype latency to courtship average: 1-2 minutes Wildtype latency to copulation average:2-5 minutes Wildtype copulation duration average 18-30 minutes

  12. Behavioral Results: Wildtype retn mutant retn control Mosaic controls Mosaics Analysis of variance concluded no behavioral difference due to genotype

  13. Behavioral results Wildtype retn mutant retn control Mosaic controls Mosaics Analysis of variance concluded no behavioral difference due to genotype

  14. Behavioral test results Wildtype retn mutant retn control Mosaic controls Mosaics Analysis of variance concluded no behavioral difference due to genotype

  15. 18 24 18 20 20 20 19 23 22 retn/cyo retn/retn 23 hthgal4/RNAi I hthgal4/RNAi III Wildtype RNAi ARID/+ RNAi I/+ hthgal4/+ hthgal4/RNAi ARID RNAi III/+ retn mutant retn control Mosaic controls Mosaics Mosaics Fertility results

  16. Reproductive tract Wildtype retn mutant

  17. retn mutant Mosaic

  18. Conclusion • Results gear us toward understanding that sterility in mosaic is due to another phenotypic mutation resulting from retn degredation • retn mutants may be sterile due to another product of retn degredation and not fully because of the loss of the common oviduct

  19. Future Work • Continue comparative work in retn reproductive tract • Use a different driver than Gal4 to increase strength of sterility • Localize retn degradation to the nervous system and not the reproductive tract

  20. One step closer to understanding the process of cell expression www.anatomy.unimelb.edu.au/.../life_cycle.jpg

  21. Acknowledgments Howard Hughes Medical Institute Dr. Barbara Taylor (Department of Zoology) Dr. Kevin Ahern (Department of Biochemistry/Biophysics) Taylor Lab

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