Lori Evans Herman Lab Institute of Molecular Biology University of Oregon SPUR Program Using Drosophila to investigate axon targeting Brain wiring is complicated ZOOM IN on: randomly labeled neurons a human brain 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 growth cone axon Each neuron extends its axon to seek its target cell body growth cone axon How can we study axon target selection? Drosophila photoreceptor axons go to different brain layers Fly eye ~ 800 ommatidia (facets) 8 7 Ommatidium 8 neurons Targets in the fly brain Drosophila photoreceptor axons go to different brain layers Fly eye ~ 800 ommatidia (facets) 8 7 Ommatidium 8 neurons Targets in the fly brain What does the fly visual system really look like? Drosophila photoreceptor axons go to different brain layers 8 7 R8 R7 R8 R7 lamina medulla Drosophila photoreceptor axons go to different brain layers 8 7 R8 R7 R8 R7 lamina medulla How can we identify genes required for R7 axons to choose the R7 target layer? We look for mutations that disrupt R7 axons R7s are m/m m/+ R8 R7 We use mosaic animals Wild type R7s 5-2 mutant R7s send axons to the wrong layer R8 R7 GFP MAb24B10 wild type R7s 5-2 mutant R7s Adult Phenotype 5-2 mutant R7s send axons to wrong layer R8 R7 GFP MAb24B10 wild type R7s 5-2 mutant R7s 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 2L 2R3L3R 5-2 mutation is on 3R chromosome or 2L 2R3L3R How can we determine where the 5-2 mutation is on the 3R chromosome? XX in XY in 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 Deficiency Kit for 3R Chromosome ? 5-2 mutant Deficiency kit with deletions of 3R chromosome used to map 5-2 Deficiency Kit for 3R Chromosome What is complementation testing? ? 5-2 mutant 5-2 failed to complement Df # mutant Df 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df #5414 dead alive dead 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df #5414 dead alive dead 5-2 mutant ? ? ? Gaps exist along the 3R chromosome 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df 5-2 mutant Df #5414 dead alive dead 5-2 mutant Gaps exist along the 3R chromosome We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr e ca sr e ca ca e ca sr e We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr e ca sr e ca ca e ca sr e We can use recombination to test whether or not this lethal mutation causes the R7 phenotype cu sr e ca sr e ca ca e ca sr e How did I create recombinant chromosomes? I set up several generations of crosses ru cu ca 5-2 TM ru cu ca ru Pr ca recomb (5-2) ru Pr ca D ; TM 3 D ; TM 6B D ; recomb (5-2) Y ; TM 6B D ; TM 3 D ; TM 6B D ; recomb (5-2) Y ; TM 6B hist D ; recomb (5-2) Y ; TM 6B hist D ; recomb (5-2) Y ; TM 6B D ; recomb (5-2) Y ; TM 6B These are the numbers of each type of recombinant chromosome I found cu sr e ca sr e ca ca e ca sr e Preliminary results have been analyzed sr e ca R8 R7 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