Transcript sim
Lab Meeting 3.23.09 Post-embryonic expression of sim Hartenstein (1993) • 3 Sim+ clusters in the larval central brain • Optic lobes D OL M V Sim • Midline of the ventral nerve cord Identified sim enhancers 1.0 2.8 3.7 Analysis of sim-GAL4 enhancer lines Transformed Fly Lines In progress 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 sim transgenic lines Construct Embryonic Expression Larval Expression 1.0 kb 2.8 kb 3.1 kb 5.5 kb 3.7 kb sandman midline cells midline glia, muscle precursors hindgut foregut, pharynx all midline cells midline glia, muscle pre. none small posterior clusters none none ventral anterior clusters (FLP out) small posterior clusters Frag 1 Frag 2 Frag 3 Frag 4 Frag 5 Intron 3 Intron 7 3’sim F1 3’sim F2 3’sim F3 3’sim F4 midline glia, muscle precursors CNS none gut CNS CNS, PNS none CNS within the next week laminar cells (OL), small post. clusters central brain NBs, GMCs none none medullar cells (OL), posterior clusters CNS none anterior lateral clusters (timeout?) Optic lobe expression sim GFP Laminar OL Neurons Medullar OL Neurons Posterior central brain clusters sim GFP Small Posterior Cluster Lateral Posterior Cluster 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 Anterior - 1.5kb Intron 3 Frag 2 Anterior Intron 3 Frag 2 - Posterior Sandman Fragment Dan’s 2.8 sandman Sim sandman Dan’s 2.8kb fragment Fragment 3 expressed in proliferating NBs Neuroblasts GMCs Post-mitotic neurons Relief of repression? sim GFP Relief of repression? 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 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? 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 P[acman] cloning of sim Two origins of replication - oriS (low copy) - oriV (high copy for injections) 1. 2. 3. 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 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 • • • • • 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? Analysis of sim-GAL4 enhancer lines Transformed Fly Lines Frag 2 Frag 3 In progress Intron 3 Intron 7 3’ sim Frag 3