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Lab Meeting 3.23.09

Lab Meeting 3.23.09. 3 Sim + clusters in the larval central brain Optic lobes Midline of the ventral nerve cord. Hartenstein (1993). D. OL. M. V. Sim. Post-embryonic expression of sim. 3.7. 2.8. Identified sim enhancers. 1.0. Transformed Fly Lines. In progress.

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Lab Meeting 3.23.09

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  1. Lab Meeting 3.23.09

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

  3. 3.7 2.8 Identified sim enhancers 1.0

  4. Transformed Fly Lines In progress 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

  5. sim transgenic lines ConstructEmbryonic ExpressionLarval Expression 1.0 kb 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 pre. small posterior clusters Frag 1 - - 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), posterior 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 within the next week

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

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

  8. Additional intronic enhancers 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 Intron 3 Frag 2 - Posterior Intron 3 Frag 2 Anterior Intron 3 Anterior - 1.5kb

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

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

  11. 1kb +Frag 3 Sim binding site Su(H) binding site Sandman + Frag 3 (4.3kb) Frag 2 Frag 3 Dan 2.8 Dan 3.1 Sandman 1kb+Frag 3 Sandman + Frag 3 Relief of repression? 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) • 500bp homology arms cloned into P[acman] vector  linearize • Retrieve fragment via gap repair between P[acman] construct and BAC clone • 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. Transformed Fly Lines In progress Analysis of sim-GAL4 enhancer lines Frag 3 Intron 3 Intron 7 Frag 2 3’ sim Frag 3

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