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 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. Map the location of cells responsible for female sterility by comparing retn flies to retn RNA-interference mosaics Goal Goal 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. = retn expression location X retained(retn) Wildtype(CSA) Mosaic

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

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

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

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

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

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

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

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

16. Wildtype retn mutant Reproductive tract

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 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 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 Continue comparative work in retn reproductive tract Use a different driver than Gal4 to increase strength of sterilityUse a different driver than Gal4 to increase strength of sterility Localize retn degradation to the nervous system and not the reproductive tract 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 Acknowledgments Howard Hughes Medical Institute Dr. Barbara Taylor (Department of Zoology) Dr. Kevin Ahern (Department of Biochemistry/Biophysics) Taylor Lab