Lab Meeting 3.23.09
Dec 18, 2015
Lab Meeting 3.23.09
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
1.0
2.8 3.7
Identified sim enhancers
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+ cells3. Further dissect fragments not restricted to central brain clusters
Transformed Fly Lines In progress
Construct Embryonic Expression Larval Expression
1.0 kb midline cells none2.8 kb midline glia, muscle precursors small posterior clusters3.1 kb hindgut none5.5 kb foregut, pharynx none3.7 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.
clustersFrag 3 CNS central brain NBs, GMCsFrag 4 none noneFrag 5 gut noneIntron 3 CNS medullar cells (OL), posterior
clustersIntron 7 CNS, PNS CNS3’sim F1 none none3’sim F2 - -3’sim F3 CNS anterior lateral clusters (timeout?)3’sim F4 within the next week
sim transgenic lines
Optic lobe expression
simGFP
Laminar OL Neurons Medullar OL Neurons
Posterior central brain clusters
Lateral Posterior Cluster
simGFP
Small Posterior Cluster
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
Sandman Fragment
Dan’s 2.8
sandman
Sim sandman Dan’s 2.8kb fragment
Fragment 3 expressed in proliferating NBs
NeuroblastsGMCsPost-mitotic neurons
Relief of repression?
simGFP
Relief of repression?
1kb +Frag 3
Sandman + Frag 3 (4.3kb)
Sim binding site
Su(H) binding site
Frag 2Frag 3
Dan 2.8Dan 3.1
Sandman1kb+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 3Is 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. 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
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?
Frag 2 Frag 3 Intron 3 Intron 7 3’ sim Frag 3
Transformed Fly Lines In progress
Analysis of sim-GAL4 enhancer lines