1. Axon Targeting and Cell Fate in the Drosophila Eye Humera Ahmad Verni Logendran Herman Lab

2. How do cells know their fate? single-cell zygote Cell division → Skin… Neurons… Muscle… Or blood cells…

3. The Drosophila eye contains different kinds of neurons

4. © National Institute for Medical Research R1-R8 express different rhodopsins and form synapses in different brain layers R1-6s express Rh1 Iris Salecker R8s express Rh5/6 R7s express Rh3/4

5. R7s must receive several signals to meet their destination 88 52 8 52 34 8 52 34 16 8 52 34 16 7 precursors undergo one more mitosis larval development

6. Taking a genetic approach to identify genes responsible for R7 development Two conventional screens using chemical mutagens: – Homozygous mutant flies – Mosaic flies New approach: Systematic removal of small regions of the Drosophila genome.

7. X Homozygous mutant flies Previous Method for Screening Chemical mutagen Dissect and examine R7 axon targeting and cell fate

8. Drawback of Screen I Only identified two genes that controlled R7 development What if genes important for R7 are also required for early development?

9. X Mosaic mutants Screen II Chemical mutagen Dissect and examine homozygous mutant R7s Heterozygous mutants

10. express FLP recombinase random mutation FRT recombination site mitotic recombination Creating Mosaic Animals Homozygous mutant Homozygous wild type R7 Parents

11. Labeling mutant cells using MARCM GFP transcription in homozygous mutant cell GFP not expressed

12. express FLP recombinase mitotic recombination Labeling mutant cells using MARCM m/m cells express GFP Homozygous mutant Heterozygous Gal80 Homozygous wild type Gal80 * All cells express Gal 4-UAS/ GFP

13. Screen II has identified new genes important to cell fate and axon targeting 1 2 3 4 5 6 7

14. Drawbacks of Screen II Laborious – Mutation must be mapped to pinpoint gene Does not cover the whole genome – Mutations are distributed randomly Not all results are significant – Wild-type phenotype

15. Solution: Our Screen! There is a library of molecularly defined deletions – Know exactly which genes the deletion removes Each deletion removes 10-50 genes

16. Solution: Our Screen! Creating stocks with specific deletions on the left arm of chromosome III that will be screened with MARCM Can systematically screen every gene in this region of the genome FRT SiteDeletion

17. + FRT SiteDeletion We attempted to recombine 55 deletions onto a FRT chromosome Meiotic recombination

18. The closer the deletion is to the FRT, the less frequently recombination occurs Recombination distance: 3 Recombination distance: 21 Recombination distance: 45

19. FRT2 Del TM6B (humoral) X FRT2 Del Meiotic recombination does not occur in males TM3 (stubble) TM6B X FRT2, Del TM3 Genetic Scheme Del TM3 FRT2 TM3 TM3 +

20. Complementation Test: Determining whether the chromosome has the deletion FRT2? Del FRT2? TM6B TM3 Del TM3 TM6 FRT2? TM3 X Del TM6B Wild-type Hu Sb Sb, Hu TM3 TM6B TM3 Del Dead Hu Sb, Hu Sb FRT2? Del TM3 X FRT2? Del Del FRT2? Del TM6B Del TM6B

21. FRT site verification Use PCR (polymerase chain reaction) to verify the presence of FRT recombination site in Drosophila’s DNA. If the FRT site is present, only then will a region specific to this site be amplified.

22. Results: Our Screen Deletion # Projected # of recombinants to be screenedActual # of recombinants scored # of recombinants with deletion # of recombinants with FRT 75637.471722 75667.471842 75647.472281 75657.4729101 75697.71600 75687.7206 2 75677.72591 75627.727113 75707.72894 75738.450N/D 75758.451922 75768.451900 75748.452154 75778.45214 0 75718.452460 75728.4530153 792111.518N/D 792211.521N/D 757811.5224 2 75831428107 7582142974 7580143043 7581143310 7584143353 792714.616 N/D 792814.63044 758714.6333 1 758816.8346 2 774519.519N/D 792919.525N/D 758919.5371 1 759119.53801 793019.538N/D 792420.617N/D v

23. Results continued… Deletion # Projected # of recombinants to be screenedActual # of recombinants scored # of recombinants with deletion # of recombinants with FRT 792620.618N/D 758620.62462 758520.6444 1 759323.24931 793326.4900 759428.349122 759655.25841 759555.269111 759774.44060 793489.82262 759989.8344 1 760189.84462 759889.849 11 760089.8516 0 760289.854 22 7607151.25210 7606151.26091 7608458.2515 0 7604>100051 11 7605>1000512 0 7729>10005900 55 deletions used for the screen 34 chromosomes (thus far) containing FRT 2 site + deletion

24. Future Directions… Use verified chromosomes to create homozygous mutant R7s Dissect retinas and brains Observe the effect in axon targeting and cell fate. Determine which gene in the deletion is required

25. Acknowledgements Herman Lab – Dr. Tory Herman, Jon Kniss, Jen Jeffress, Adam Miller, Eric Lyons, Scott Holbrook Peter O’Day SPUR

26. Questions?