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