Christine Saseun Doe Lab University of Oregon

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Presentation transcript:

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/ TM6C56 X w-/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 O’Day Beth Roy SPUR Participants University of Oregon