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Stephanie Freer Lab Meeting 7.6.09

Stephanie Freer Lab Meeting 7.6.09. 3 anterior Sim + clusters Optic lobes Midline of the VNC Posterior clusters. Hartenstein (1993). Post-embryonic expression of sim. D. Posterior. OL. B. T. Sim. Anterior. Analysis of sim- GAL4 enhancer lines.

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Stephanie Freer Lab Meeting 7.6.09

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  1. Stephanie Freer Lab Meeting 7.6.09

  2. 3 anterior Sim+ clusters Optic lobes Midline of the VNC Posterior clusters Hartenstein (1993) Post-embryonic expression of sim D Posterior OL B T Sim Anterior

  3. Analysis of sim-GAL4 enhancer lines To visualize the specific projection pattern for each enhancer-GAL4 line: 1. Cross enhancer-GAL4 to UAS-GFP.nls or UAS tau-GFP 2. Analyze embryonic and larval brains for GFP-expression in sim+cells 3. Further dissect fragments not restricted to central brain clusters

  4. sim transgenic lines ConstructEmbryonic ExpressionLarval Expression 1.0 kb all midline cells none 2.8 kb midline glia, muscle precursors small posterior clusters 3.1 kb hindgut none 5.5 kb foregut, pharynx none 3.7 kb all midline cells ventral anterior clusters (FLP out) sandman midline glia, muscle precursors small posterior clusters Frag 1 coming soon - Frag 2 midline glia, muscle precursors laminar cells (OL), small post. clusters Frag 3 CNS central brain NBs, GMCs Frag 4 none none Frag 5 gut none Intron 3 CNS medullar cells (OL), ant./post. clusters Intron 7 CNS, PNS CNS 3’sim F1 none none 3’sim F2 - - 3’sim F3 CNS anterior lateral clusters (timeout?) 3’sim F4 none none

  5. sim GFP Optic lobe expression Laminar OL Neurons Medullar OL Neurons

  6. Posterior central brain clusters sim GFP Small Posterior Cluster Lateral Posterior Cluster

  7. Expression in the anterior clusters BP106 GFP Intron 3-GAL4 x UAS mCD8-GFP Intron 3 contains enhancer elements responsible for sim expression in all 3 of the anterior central brain clusters

  8. Intron 3 Frag 3 Intron 3 Frag 2 Anterior Additional intronic enhancers 3 Intron 3 500bp fragments Intron 3 Frag 1 – posterior clusters - subset of anterior clusters? - optic lobes Intron 3 Frag 2 – no expression Intron 3 Frag 3 – Optic lobes (medullar subset) New fragment spanning intron 4-6 to be injected

  9. Intron 3 F1-GAL4 x UAS mCD8-GFP nc82 GFP Posterior Anterior GFP expression - a subset of anterior central brain cells - medullar optic lobe expression (subset) - posterior clusters

  10. simJ1-47 WT Reduction of late sim leads to structural defects in the adult • simJ1-47 - temperature sensitive allele • Neuropil disorganization in the adult • Sterile with behavioral defects Rein et al., 2002

  11. Central complex structure Neckameyer and Matsuo, 2008 Fan-shaped body – visual pattern learning Ellipsoid body, noduli (ebo) – decay in walking activity Protocerebral bridge (nob) – deficits in step length Abnormal turning behavior, “left-right bargaining” in mutants of both eb and pb  defects typically at the midline

  12. nc82 GFP Intron 3-GAL4 expression in the adult brain Expression in a subset of fan-shaped body axons Lack of expression in the protocerebral bridge and ellipsoid body In general, lots of axons stained  specificity?

  13. Intron 3-GAL4 expression in the optic lobe nc82 GFP

  14. Expression in the SOG nc82 GFP Intron 3 F1-GAL4 x UAS mCD8-GFP Expression in the anterior subeosphogeal ganglion, send projections dorsally Corresponds to Trdm?

  15. nc82 GFP Summary of adult expression Intron 3 F1-GAL4 x UAS mCD8-GFP Expression in the SOG, optic lobes, PNs in the AL, fan-shaped body, MBs, superior medial protocerebrum, superior lateral protocerebrum

  16. Further dissection of Intron 3 1.4kb F1 F3 F2 F1a F1b F1+ F2 • Separate optic lobe expression from central brain expression • Capture all central brain neurons within one line • Another possibility: Mutate potential optic lobe elements

  17. Transmitter phenotype of sim-expressing midline cells in the embryo * * * Sim VGlut/Sim En/Sim Gad1/Sim

  18. Posterior VGlut expression VGlut Sim VGlut RNA co-localizes with Sim protein Neurotransmitter phenotype in posterior sim-positive neurons  VGlut

  19. Sim Sim En En intron 1 intron 2 En En En En Sim Sim intron 3 CG3557 4.8 kb 2.8 kb Frag 8 Frag 1 2.5 kb Frag 2 3.7 kb Frag 4 2.7 kb Frag 5 0.8 kb 0.6 kb Frag 7 Frag 9 2.4 kb Frag 6 VGlut Fragment 4-6 in the 3rd instar brain Sim GFP

  20. VGlut Antibody in 3rd Instar Larvae VGlut Tau No apparent protein co-localization at the cell bodies  VGlut protein in transit to the synapse VGlut staining absent in secondary lineages labeled by BP106 (Daniels et al. 2008)

  21. Transmitter phenotype in anterior sim-positive central brain clusters • 1-2 VGlut+, sim+ cells observed for each cluster • No Gad1+, sim+ cells observed Two options: 1. Cells not differentiated yet  Stain pupal and adult brains 2. Another transmitter involved - monoamines: dopamine, serotonin, octopamine - neuropeptides: neuropeptide F Medial Cluster Sim Gad1 VGlut

  22. Summary • Analyze additional fragments - Frag 1, other introns, 3’ sim F2 - Intron 3 dissection • Neurotransmitter staining in pupae and adults • Behavioral Analysis - Larval and adult locomotion Post-embryonic expression within Intron 3 Expression from intron 3-GAL4 in the adult - Subset in the fan-shaped body - Structures other than the CC Posterior clusters are glutamatergic

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