Christine Saseun Doe Lab University of Oregon
Generating tools for Studying Asymmetric Cell Division and Brain Morphogenesis in Drosophila Melanogaster Christine Saseun Doe Lab University of Oregon Drosophila as a Model Organism
Genetics: Huge collection of mutant strains available. Entire genome sequenced Handling: Rapid development & short life cycles Tools: Mutations induced easily & Large scale crosses can be set up and followed through several generations Larval brains Cell types in larval brains: Neuroblasts, GMCs(ganglion mother cells), Glia, and Neurons Structure of the Brain: Projects Question: How to study mutant phenotypes in individual brain lineages/cell types? Aim: Labeling individual lineages/cell types. Experiments: GAL4 Screens Clonal Analysis 1. GAL 4 Screens GAL4 system Brand A., Perrimon N. 1. GAL 4 Screens Cross for the experiment:
Enhancer TRAP GAL 4 x UAS_GFP GFP (+) Larvae were chosen, dissected, and fixed. And were later imaged on a confocal microscope. A62 Ubiquitous A221 Glia Specific A217 Lineage Specific Lineage/cell type specific
070 Lineage/cell type specific GAL 4 Lines Ubiquitous/no expression Glia specific
Lineage/cell type specific 12 6 14 2. Clonal Analysis MARCM ( mosaic analysis with a repressible cell marker) Aging and Heat shocking regime Age (hrs.) NB Clones Glia clones 2-6h 4-8h 6-10h 8- 12h 12-16h 14-18h 18-22h Lee T., Luo L. 2. Clonal Analysis P.hs FLP/hs FLP; Tub Gal 80 FRT40A/ CyO; Tub Gal 4_UAS CD8::GFP/ TM6C56Xw-/Y ; FRT40A/ FRT 40A; +/ + F1. hs FLP/ w-; Tub Gal 80 FRT40A/ FRT 40A; Tub Gal 4_UAS CD8:: GFP/+ OR hs FLP/Y ; Tub Gal80 FRT40A/ FRT40A; Tub Gal 4_UAS CD8:: GFP/+ Anti-body staining with: Dpn: Neuroblast Repo: Glia GFP: Membrane/clone Glia + Neuroblast clones
Repo (+) Glia Dpn (+) Neuroblast GFP (+) Membrane Neuroblast clones only
Repo (+) Glia Dpn (+) Neuroblast GFP (+) Membrane Glia clones only Repo (+) Glia Dpn (+) Neuroblast GFP (+) Membrane Clonal Analysis Age (hrs.) NB Clones Glia clones 2-6h 4.25 X 4-8h 2
3.375 8- 12h 7.5 12-16h 5 14-18h 3.5 18-22h 9.5 Conclusions Lineage & cell-type specificity can be achieved using different Gal4 lines and clonal analysis. Certain GAL 4 lines are ideal for different lineages and can be combined with the MARCM technique to produce an effective tool for studying brain morphogenesis. Acknowledgements Chris Doe Clemens Cabernard Doe Lab Peter ODay
Beth Roy SPUR Participants University of Oregon