1. Stephanie Freer Lab Meeting 7.6.09

2. Post-embryonic expression of sim 3 anterior Sim + clusters Optic lobes Midline of the VNC Posterior clusters Hartenstein (1993) Sim OL D B T Posterior 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. Construct Embryonic ExpressionLarval Expression 1.0 kb all midline cellsnone 2.8 kb midline glia, muscle precursors small posterior clusters 3.1 kb hindgutnone 5.5 kb foregut, pharynxnone 3.7 kb all midline cellsventral anterior clusters (FLP out) sandman midline glia, muscle precursorssmall posterior clusters Frag 1 coming soon - Frag 2 midline glia, muscle precursorslaminar cells (OL), small post. clusters Frag 3 CNScentral brain NBs, GMCs Frag 4 nonenone Frag 5 gutnone Intron 3 CNSmedullar cells (OL), ant./post. clusters Intron 7 CNS, PNSCNS 3’sim F1 nonenone 3’sim F2 -- 3’sim F3 CNSanterior lateral clusters (timeout?) 3’sim F4 nonenone sim transgenic lines

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

6. Posterior central brain clusters Lateral Posterior Cluster sim GFP Small 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. Additional intronic enhancers Intron 3 Frag 2 Anterior 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 Intron 3 Frag 3

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

10. Reduction of late sim leads to structural defects in the adult sim J1-47 - temperature sensitive allele Neuropil disorganization in the adult Sterile with behavioral defects WT sim J1-47 Rein et al., 2002

11. Neckameyer and Matsuo, 2008 Central complex structure 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. Intron 3-GAL4 expression in the adult brain nc82 GFP 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. Summary of adult expression nc82 GFP 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 F2 F3 F1a F1b F1+ F2 1.Separate optic lobe expression from central brain expression 2.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 En/Sim VGlut/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. VGlut Fragment 4-6 in the 3 rd instar brain Sim GFP En Sim CG3557 2.4 kb 2.5 kb 2.8 kb 3.7 kb 2.7 kb 4.8 kb 0.8 kb 0.6 kb Frag 2 Frag 8 Frag 4 Frag 1 Frag 6 Frag 5 Frag 9 Frag 7 intron 1 intron 3 intron 2

20. VGlut Antibody in 3 rd 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 Sim Gad1 VGlut Medial Cluster

22. Summary 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 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