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Axon Targeting and Cell Fate in the Drosophila Eye Humera Ahmad Verni Logendran Herman Lab How do cells know their fate? Neurons… Cell division → Skin… single-cell zygote Muscle… Or blood cells… The Drosophila eye contains different kinds of neurons R1-R8 express different rhodopsins and form synapses in different brain layers R1-6s express Rh1 R8s express Rh5/6 R7s express Rh3/4 Iris Salecker © National Institute for Medical Research R7s must receive several signals to meet their destination larval development 8 2 8 5 1 6 8 2 5 3 4 2 8 5 3 4 precursors undergo one more mitosis 1 7 6 2 8 5 3 4 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. Previous Method for Screening X Homozygous mutant flies Dissect and examine R7 axon targeting and cell fate Chemical mutagen Drawback of Screen I Only identified two genes that controlled R7 development What if genes important for R7 are also required for early development? Screen II X Heterozygous mutants Mosaic mutants Dissect and examine homozygous mutant R7s Chemical mutagen Creating Mosaic Animals FRT recombination site random mutation express FLP recombinase Homozygous mutant Homozygous wild type mitotic recombination R7 Parents Labeling mutant cells using MARCM GFP transcription in homozygous mutant cell GFP not expressed Labeling mutant cells using MARCM m/m cells express GFP express FLP recombinase Homozygous mutant Gal80 Homozygous wild type Gal80 Gal80 Gal80 mitotic recombination * All cells express Gal 4-UAS/ GFP Gal80 Heterozygous Gal80 Screen II has identified new genes important to cell fate and axon targeting 3 4 2 1 7 5 6 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 Solution: Our Screen! • There is a library of molecularly defined deletions – Know exactly which genes the deletion removes • Each deletion removes 10-50 genes 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 Deletion FRT Site We attempted to recombine 55 deletions onto a FRT chromosome FRT Site Deletion + Meiotic recombination The closer the deletion is to the FRT, the less frequently recombination occurs Recombination distance: 45 Recombination distance: 21 Recombination distance: 3 Genetic Scheme FRT2 X FRT2 Del TM6B (humoral) Meiotic recombination does not occur in males FRT2 X TM3 (stubble) Del TM6B FRT2 Del FRT2 , Del TM3 TM3 TM3 + TM3 Complementation Test: Determining whether the chromosome has the deletion FRT2? X TM3 FRT2? Del FRT2? Del FRT2? Del FRT2? Del Wild-type Hu FRT2? Del TM6B TM3 TM3 Del TM6 Sb, Hu Dead Hu Sb, Hu TM6B TM3 TM3 TM6B Del TM6B Sb X TM3 TM6B Del Del Sb 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. Results: Our Screen Deletion # 7563 Projected # of recombinants to be screened 7.47 Actual # of recombinants scored 17 # of recombinants with deletion 2 # of recombinants with FRT 2 7566 7564 7565 7.47 7.47 7.47 18 22 29 4 8 10 2 1 1 7569 7568 7567 7562 7570 7573 7575 7576 7574 7577 7571 7572 7921 7922 7578 7583 7582 7580 7581 7584 7927 7928 7587 7588 7745 7929 7589 7591 7930 7924 7.7 7.7 7.7 7.7 7.7 8.45 8.45 8.45 8.45 8.45 8.45 8.45 11.5 11.5 11.5 14 14 14 14 14 14.6 14.6 14.6 16.8 19.5 19.5 19.5 19.5 19.5 20.6 16 20 25 27 28 0 19 19 21 21 24 30 18 21 22 28 29 30 33 33 16 30 33 34 19 25 37 38 38 17 0 6 9 11 9 N/D 2 0 5 4 6 15 N/D N/D 4 10 7 4 1 5 N/D 4 3 6 N/D N/D 1 0 N/D N/D 0 2 1 3 4 N/D 2 0 4 0 0 3 N/D N/D 2 7 4 3 0 3 N/D 4 1 2 N/D N/D 1 1 N/D N/D v Results continued… Deletion # Projected # of recombinants to be screened Actual # of recombinants scored # of recombinants with # of recombinants with deletion FRT 7926 20.6 18 N/D N/D 7586 20.6 24 6 2 7585 20.6 44 4 1 7593 23.2 49 3 1 7933 26.4 9 0 0 7594 28.3 49 12 2 7596 55.2 58 4 1 7595 55.2 69 11 1 7597 74.4 40 6 0 7934 7599 89.8 89.8 22 34 6 4 2 1 7601 89.8 44 6 2 7598 89.8 49 1 1 7600 89.8 51 6 0 7602 89.8 54 2 2 7607 151.2 52 1 0 7606 151.2 60 9 1 7608 458.2 51 5 0 7604 >1000 51 1 1 7605 >1000 51 2 0 7729 >1000 59 0 0 • 55 deletions used for the screen • 34 chromosomes (thus far) containing FRT 2 site + deletion 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 Acknowledgements • Herman Lab – Dr. Tory Herman, Jon Kniss, Jen Jeffress, Adam Miller, Eric Lyons, Scott Holbrook • Peter O’Day • SPUR Questions?