1. Lab Meeting 3.23.09

2. Post-embryonic expression of sim 3 Sim + clusters in the larval central brain Optic lobes Midline of the ventral nerve cord Hartenstein (1993) Sim OL D M V

3. 1.0 2.8 3.7 Identified sim enhancers

4. 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 Transformed Fly LinesIn progress

5. Construct Embryonic ExpressionLarval Expression 1.0 kb 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 pre.small posterior clusters Frag 1 - - 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), posterior clusters Intron 7 CNS, PNSCNS 3’sim F1 nonenone 3’sim F2 -- 3’sim F3 CNSanterior lateral clusters (timeout?) 3’sim F4 within the next week sim transgenic lines

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

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

8. Additional intronic enhancers Intron 3 Anterior - 1.5kb Intron 3 Frag 2 Anterior Intron 3 Frag 2 - Posterior 3 Intron 3 500bp fragments Intron 3 Frag 1 – no expression Intron 3 Frag 2 – posterior clusters - glial cells, unspecific? Intron 3 Frag 3 – Optic lobes (medullar) New fragment spanning intron 4-6 to be injected

9. Sandman Fragment Dan’s 2.8 sandman Sim sandman Dan’s 2.8kb fragment

10. Fragment 3 expressed in proliferating NBs Neuroblasts GMCs Post-mitotic neurons Relief of repression? sim GFP

11. Relief of repression? 1kb +Frag 3 Sandman + Frag 3 (4.3kb) Sim binding site Su(H) binding site Frag 2 Frag 3 Dan 2.8 Dan 3.1 Sandman 1kb+Frag 3 Sandman + Frag 3 Dan’s 3.1 - no larval expression, 3 Su(H) 1kb+Frag3 – includes only one Su(H), also in Frag 3 Is sandman fragment involved?

12. P[acman] cloning of sim Recombineering mediated cloning Sequence from BAC clones Fragments up to 133 kb ΦC31 compatible Allows for the cloning of the entire sim locus, large subregions

13. P[acman] cloning of sim Two origins of replication - oriS (low copy) - oriV (high copy for injections) 1.500bp homology arms cloned into P[acman] vector  linearize 2.Retrieve fragment via gap repair between P[acman] construct and BAC clone 3.Induce high copy number for injection

14. P[acman] cloning of sim 500bp 16.4 kb 16.7 kb 32.6 kb Why? Narrow down region responsible for expression - If multiple distant elements are required for expression - if elements were separated, chopped up

15. Analyze additional fragments p[acman] transgenic lines FLP out experiments Better characterize 1.0kb fragment What embryonic NBs correspond to sim positive central brain clusters? When are they born?

16. Frag 2 Frag 3Intron 3Intron 7 3’ sim Frag 3 Transformed Fly LinesIn progress Analysis of sim-GAL4 enhancer lines