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Using Drosophila to investigate axon targeting Lori Evans Herman Lab Institute of Molecular Biology University of Oregon SPUR Program Brain wiring is complicated a human brain ZOOM IN on: randomly labeled neurons Brain wiring is organized into layers a human brain Brain wiring is organized into layers a human brain How do neurons grow and connect during development? Each neuron extends its axon to seek its target cell body axon growth cone Each neuron extends its axon to seek its target cell body axon growth cone How can we study axon target selection? Drosophila photoreceptor axons go to different brain layers Fly eye ~ 800 ommatidia (facets) Ommatidium 8 neurons 7 8 Targets in the fly brain Drosophila photoreceptor axons go to different brain layers Fly eye ~ 800 ommatidia (facets) Ommatidium 8 neurons 7 8 Targets in the fly brain What does the fly visual system really look like? Drosophila photoreceptor axons go to different brain layers 7 8 lamina R8 medulla R8 R7 R7 Drosophila photoreceptor axons go to different brain layers 7 8 R8 lamina R7 medulla R8 R7 How can we identify genes required for R7 axons to choose the R7 target layer? We look for mutations that disrupt R7 axons Wild type R7s We use mosaic animals R7s are m/m m/+ R8 R7 5-2 mutant R7s send axons to the wrong layer 5-2 mutant R7s wild type R7s MAb24B10 GFP R8 R7 Adult Phenotype 5-2 mutant R7s send axons to wrong layer wild type R7s 5-2 mutant R7s MAb24B10 GFP R8 R7 My main question of interest: What gene does the 5-2 mutation disrupt? 5-2 mutation is on 3R chromosome XX in ♀ XY in ♂ or 1 2 2L 2R 3 4 3L 3R 5-2 mutation is on 3R chromosome XX in ♀ XY in ♂ or 1 2 2L 2R 3 3L 3R 4 How can we determine where the 5-2 mutation is on the 3R chromosome? We used two methods to map the 5-2 mutation • Deficiency Mapping • Recombination Mapping Deficiency kit with deletions of 3R chromosome used to map 5-2 5-2 mutant ? Deficiency Kit for 3R Chromosome Deficiency kit with deletions of 3R chromosome used to map 5-2 5-2 mutant ? Deficiency Kit for 3R Chromosome What is complementation testing? 5-2 failed to complement Df #5414 5-2 mutant 5-2 mutant 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df #5414 dead alive alive alive dead Gaps exist along the 3R chromosome 5-2 mutant 5-2 mutant dead 5-2 mutant Df ? alive 5-2 mutant Df ? alive 5-2 mutant Df ? alive 5-2 mutant Df #5414 dead Gaps exist along the 3R chromosome 5-2 mutant 5-2 mutant dead 5-2 mutant Df alive 5-2 mutant Df alive 5-2 mutant Df alive 5-2 mutant Df #5414 dead We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr sr e ca e ca e ca ca sr e We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr sr e ca e ca e ca ca sr e We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr sr e ca e ca e ca ca sr e How did I create recombinant chromosomes? ♀ 5-2 ru cu ca TM 3 ru cu ca ♀ ♂ 5-2 ru cu ca ru cu ca ru Pr ca recomb (5-2) ♂ ♂ D ; TM 3 ru Pr ca D ; TM 6B D ; recomb (5-2) ♀ I set up several generations of crosses ♂ Y ; TM 6B ♂ D ; recomb (5-2) ♂ Y ; TM 6B D ; TM 3 D ; recomb (5-2) hist ♀ Y ; TM 6B ♀ D ; TM 6B D ; recomb (5-2) D ; recomb (5-2) Y ; TM 6B ♂ Y ; TM 6B hist ♂ These are the numbers of each type of recombinant chromosome I found cu sr sr e e ca ca ca 32 63 e 50 ca sr e 10 Preliminary results have been analyzed sr R8 R7 e ca Future Work Hypothesis Correct • Identify gene – Use smaller deficiencies Hypothesis Incorrect • Look at other recombinants • Find 5-2 mutation causing phenotype • Identify gene Conclusion The 5-2 gene identified to be involved in axon target selection in Drosophila may also play a role in axon target selection in humans. Acknowledgments Herman Lab Tory Herman Adam Miller Eric Lyons Jennifer Jeffress Heather Seymour Jon Kniss Scott Holbrook Stefan Buetow *Lola, Bert, Bode, Jobie* SPUR Peter O’ Day Beth Roy SPUR Participants SPUR Funders